Antibodies to Symmetrically Dimethylated Arginine Analytes and Use Thereof

ABSTRACT

Disclosed is an antibody which binds to a symmetrically dimethylated arginine analyte that can be used to detect a symmetrically dimethylated arginine analyte in a sample, such as in a homogeneous enzyme immunoassay method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority pursuant to 35 U.S.C. § 119(e) to thefiling date of U.S. Provisional Application Ser. No. 62/828,769 filed onApr. 3, 2019, the disclosure of which is herein incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to the field of immunoassays, and inparticular to antibodies which bind to a symmetrically dimethylatedarginine analyte that can be used in immunoassays for detection of asymmetrically dimethylated arginine analyte.

INTRODUCTION

Chronic kidney disease (CKD) is a common problem in dogs and cats. Thereis no cure available for CKD, but medical management is available forpatients with this disease. Research has focused on earlier detection ofCKD with the goal of instituting medical management and monitoring asearly in the disease course as possible. Symmetric dimethylarginine hasrecently emerged as a novel renal excretory biomarker that may aid inearly detection of CKD in cats and dogs.

Symmetric dimethylarginine (SDMA; FIG. 1), in addition to asymmetricdimethylarginine (ADMA; FIG. 2) and monomethylarginine (MMA, orN-methylarginine; FIG. 2) are derived from posttranslationalmodification (methylation) of proteins containing arginine residueswithin almost every cell. After proteolysis, or protein breakdown, theseprotein residues are released into the circulation. The potential use ofserum symmetric dimethylarginine as a marker for evaluation of kidneydisease, cardiovascular health, atherosclerosis, rheumatoid arthritis,and other diseases, has been extensively studied.

Symmetric dimethylarginine is eliminated from the body primarily viarenal excretion. Unlike creatinine, symmetric dimethylarginine isunaffected by non-renal factors including lean body mass. Symmetricdimethylarginine does not bind to protein, unlike asymmetricdimethylarginine, which is partially bound to plasma proteins.Correlations between asymmetric dimethylarginine and glomerularfiltration rate (GFR) have been shown to be much weaker, likely due tothe protein-bound fraction of asymmetric dimethylarginine, which hindersglomerular filtration.

Serum concentrations of symmetric dimethylarginine are also increased inhuman patients with CKD. It has been shown that serum symmetricdimethylarginine concentrations are inversely correlated with GFR.

A circulating metabolite of symmetric dimethylarginine and symmetricNα-acetyl-dimethylarginine (FIG. 1) has also been identified and it mayalso correlate with GRF. The acetylation reaction of symmetricdimethylarginine occurs outside the cell resulting in the metabolite.

Because symmetric dimethylarginine is almost exclusively eliminated bythe kidneys, this makes it an ideal candidate for a GFR biomarker. Thus,symmetric dimethylarginine is an emerging endogenous biomarker of kidneyfunction that is already widely used in veterinary medicine.

Enzyme-linked immunosorbent assay (ELISA) to quantify symmetricdimethylarginine is commercially available. These methods involve asample preparation step that includes the addition of an acylatingderivatizing reagent to a sample suspected of containing symmetricaldimethylarginine prior to quantifying symmetric dimethylarginine.Symmetric dimethylarginine is quantitatively converted into Nα-acyl-SDMAby the acylation reagent. The antibody used in the ELISA product bindsonly the Nα-acylated form of symmetric dimethylarginine and not to freesymmetric dimethylarginine.

More recently, a competitive immunoassay has been developed for freesymmetric dimethylarginine using antibodies that detect only freesymmetric dimethylarginine but do not cross-react with Nα-acylatedsymmetric dimethylarginine. The antibodies used in the competitiveimmunoassay are developed against a particular immunogen that produceantibodies specific to only free symmetric dimethylarginine. Haptenswere used that are derivatives involving the chemical modification ofthe acidic carboxyl group (—COOH) of symmetric dimethylarginine.Antibodies were used to detect free symmetric dimethylarginine (i.e.,symmetric dimethylarginine not part of a polypeptide chain) and show noor substantially no cross-reactivity with asymmetric dimethylarginine,L-arginine, and N-methylarginine.

Measurement of symmetric dimethylarginine can also be performed usingliquid chromatography tandem mass spectrometry (LC-MS/MS) instruments.LC-MS/MS technology remains complex and requires a significant level ofexpertise for test development and operation. In addition, LC-MS/MSassays are not fully automated and require significant samplepreparation and time before a result can be produced, increasing itsturnaround time in comparison with automated assays for serumcreatinine.

There is an ongoing need to develop tools that will enable detection ofsymmetrically dimethylated arginine analytes.

SUMMARY

The present disclosure provides methods for immunoassay of asymmetrically dimethylated arginine analyte. In particular, the presentdisclosure relates to the use of derivatives of symmetricaldimethylarginine in a signal producing immunoassay system. The presentdisclosure also relates to the use of immunogens of symmetricaldimethylarginine used for producing antibodies for capture of suchanalytes. As used herein, the term “symmetrically dimethylated arginineanalyte” refers to analytes having an antibody binding epitope which iscommon to symmetric dimethylarginine. Analytes included in thesymmetrically dimethylated arginine analytes include symmetricdimethylarginine (SDMA), symmetric Nα-acylated-dimethylarginine, such assymmetric Nα-acetyl-dimethylarginine, and SDMA-peptide derivatives, suchas SDMA-Gly-Gly dipeptide.

In some embodiments, the present disclosure provides symmetricdimethylarginine haptens alkylated on the nitrogen atom of the α-aminogroup of symmetric dimethylarginine (FIG. 1). In certain embodiments,such haptens are used to produce antibodies specific for symmetricallydimethylated arginine analytes.

In some embodiments, the present disclosure provides symmetricdimethylarginine derivatives acylated on the nitrogen atom of theα-amino group of symmetric dimethylarginine (FIG. 1). In certainembodiments, such derivatives are used to produce conjugates useful inthe immunoassays described herein.

In some embodiments, the present disclosure provides a polyclonal ormonoclonal antibody that specifically binds to a symmetricdimethylarginine metabolite, such as symmetricNα-acetyl-dimethylarginine (FIG. 1).

In some embodiments, the present disclosure provides a polyclonal ormonoclonal antibody that specifically binds to free symmetricdimethylarginine, and symmetric Nα-acetylated-dimethylarginine protein(FIG. 1).

In some embodiments, the antibody may specifically bind to one or moreof asymmetric dimethylarginine, L-arginine, and N-methylarginine (FIG.2).

In some embodiments, the present disclosure provides a polyclonal ormonoclonal antibody that specifically binds to free symmetricdimethylarginine and a SDMA-peptide derivative, such as SDMA-Gly-Glydipeptide (FIG. 3).

In some embodiments, the present disclosure provides methods for thesyntheses of symmetrical dimethylarginine haptens, immunogens andconjugates starting from symmetrical dimethylarginine. In someembodiments, the synthesis includes coupling through the nitrogen atomof the α-amino group of symmetrical dimethylarginine with a linkinggroup to a protein or a label (e.g., a label enzyme).

An example of an embodiment of the present disclosure is a compound ofFormula 1 shown below:

wherein:

R¹ is —Y—Z; and

Y is a linking group and Z is selected from hydrogen, OH, SH, S-acyl,O-alkyl, halogen, NH₂, epoxy, maleimidyl, haloacetamide, carboxyl,activated carboxyl, an immunogenic carrier, a protein, and a label.

Aspects of the present disclosure include antibodies. In someembodiments, an antibody of the present disclosure specifically binds toany of the compounds of the present disclosure, including any of thecompounds of Formula 1 described elsewhere herein.

Nucleic acids that encode any of the antibodies of the presentdisclosure are also provided, as are expression vectors comprising suchnucleic acids, and cells comprising such nucleic acids and expressionvectors.

Also provided are methods of making the antibodies of the presentdisclosure. The methods include culturing a cell of the presentdisclosure under conditions suitable for the cell to express theantibody, wherein the antibody is produced.

Aspects of the present disclosure further include compositions. Acomposition of the present disclosure may include any of the antibodies,nucleic acids, expression vectors, and/or cells of the presentdisclosure.

Also provided are methods for determining an amount of at least onesymmetrically dimethylated arginine analyte in a medium. In certainembodiments, such methods include combining in a medium a samplesuspected of containing at least one symmetrically dimethylated arginineanalyte, and an antibody of the present disclosure. Such methods furtherinclude determining the presence or absence of a complex comprising thesymmetrically dimethylated arginine analyte and the antibody, whereinthe presence of the complex indicates the presence of the symmetricallydimethylated arginine analyte in the sample.

Aspects of the present disclosure further include kits. According tosome embodiments, the kits find use in determining an amount of at leastone symmetrically dimethylated arginine analyte in a sample. In certainembodiments, a kit of the present disclosure includes any of theantibodies of the present disclosure, and instructions for using theantibody to determine an amount of at least one symmetricallydimethylated arginine analyte in a sample. Such kits may further includeany of the compounds of Formula 1 of the present disclosure. Accordingto some embodiments, a kit of the present disclosure includes any of thecompounds of Formula 1 of the present disclosure, and instructions forusing the compound to determine an amount of at least one symmetricallydimethylated arginine analyte in a sample. Such kits may further includeany of the antibodies of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will be more fully understood from the followingdetailed description thereof taken in connection with the accompanyingdrawings which form a part of this application and in which:

FIG. 1 shows the chemical structures for symmetric dimethylarginine andsymmetric Nα-acetyl-dimethylarginine.

FIG. 2 shows the chemical structures for asymmetric dimethylarginine,monomethylarginine and arginine.

FIG. 3 shows chemical structures for an SDMA-Gly-Gly dipeptide.

FIG. 4 (left) shows the SDMA-M (Nα-alkyl-SDMA) hapten which is modifiedon the α-amino nitrogen atom of symmetric dimethylarginine, according toembodiments of the present disclosure. FIG. 4 (right) shows theSDMA-SBAP (Nα-acyl-SDMA) hapten which is modified on the α-aminonitrogen atom of symmetric dimethylarginine, according to embodiments ofthe present disclosure.

FIG. 5 shows the synthesis scheme for the SDMA-M (Nα-alkyl-SDMA) hapten,according to embodiments of the present disclosure.

FIG. 6 shows the synthesis scheme for the SDMA-SBAP (Nα-acyl-SDMA)hapten, according to embodiments of the present disclosure.

FIG. 7 shows the synthesis scheme for SDMA-M-SH-G6PDH, according toembodiments of the present disclosure.

FIG. 8 shows the synthesis scheme for the SDMA-M-SH-KLH immunogen,according to embodiments of the present disclosure.

FIG. 9 shows the synthesis scheme for the SDMA-SBAP-SH-BSA immunogen,according to embodiments of the present disclosure.

FIG. 10 shows an antibody screening technique, according to embodimentsof the present disclosure.

FIG. 11 shows a graph of a symmetric dimethylarginine calibration curveusing rabbit polyclonal antibody #26494, monoclonal antibody 1H2/1K4,monoclonal antibody 3H1/3K3, and monoclonal antibody 5H1/5K1, accordingto embodiments of the present disclosure.

FIG. 12 shows a graph of a symmetric dimethylarginine calibration curveusing rabbit polyclonal antibody #21342, monoclonal antibody 1H4/1K4,monoclonal antibody 8H1/8K3, and monoclonal antibody 18H1/18K2,according to embodiments of the present disclosure.

FIG. 13 shows a graph of a symmetric Nα-acetyl-dimethylargininecalibration curve using rabbit polyclonal antibody #26494, monoclonalantibody 1H2/1K4, monoclonal antibody 3H1/3K3, monoclonal antibody5H1/5K1, according to embodiments of the present disclosure.

FIG. 14 shows a graph of a symmetric Nα-acetyl-dimethylargininecalibration curve using rabbit polyclonal antibody #21342, monoclonalantibody 1H4/1K4, monoclonal antibody 8H1/8K3, and monoclonal antibody18H1/18K2, according to embodiments of the present disclosure.

FIG. 15 shows graphs of spike-recovery experiments of symmetricdimethylarginine using rabbit polyclonal antibody #26494 (bottom right),monoclonal antibody 1H2/1K4 (top left), monoclonal antibody 3H1/3K3 (topright), and monoclonal antibody 5H1/5K1 (bottom left), according toembodiments of the present disclosure.

FIG. 16 shows graphs of spike-recovery experiments of symmetricdimethylarginine using rabbit polyclonal antibody #21342 (bottom right),monoclonal antibody 1H4/1K4 (top left), monoclonal antibody 8H1/8K3 (topright), and monoclonal antibody 18H1/18K2 (bottom left), according toembodiments of the present disclosure.

FIG. 17 shows graphs of spike-recovery experiments of symmetricNα-acetyl-dimethylarginine using rabbit polyclonal antibody #26494(bottom right), monoclonal antibody 1H2/1K4 (top left), monoclonalantibody 3H1/3K3 (top right), monoclonal antibody 5H1/5K1 (bottom left),according to embodiments of the present disclosure.

FIG. 18 shows graphs of spike-recovery experiments of symmetricNα-acetyl-dimethylarginine using rabbit polyclonal antibody #21342(bottom right), monoclonal antibody 1H4/1K4 (top left), monoclonalantibody 8H1/8K3 (top right), and monoclonal antibody 18H1/18K2 (bottomleft), according to embodiments of the present disclosure.

FIG. 19 shows graphs of spike-recovery experiments of SDMA-Gly-Glydipeptide using monoclonal antibody 3H1/3K3 (left) and monoclonalantibody 8H1/8K3 (right), according to embodiments of the presentdisclosure.

FIG. 20 shows a graph of spike-recovery experiments of SDMA andsymmetric Nα-acetyl-dimethylarginine using rabbit polyclonal antibody#27410, according to embodiments of the present disclosure.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed, to the extent that suchcombinations embrace subject matter that are, for example, compoundsthat are stable compounds (i.e., compounds that can be made, isolated,characterized, and tested for biological activity). In addition, allsub-combinations of the various embodiments and elements thereof (e.g.,elements of the chemical groups listed in the embodiments describingsuch variables) are also specifically embraced by the present inventionand are disclosed herein just as if each and every such sub-combinationwas individually and explicitly disclosed herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

Before proceeding further with the description of the specificembodiments of the present disclosure, a number of terms will bedefined.

Definitions

Analyte

A compound or composition to be measured, the material of interest. Theanalyte is a member of a specific binding pair (sbp) and may be aligand, which is mono- or polyvalent, usually antigenic or haptenic, andis a single compound or plurality of compounds which share at least onecommon epitopic or determinant site.

Sample Suspected of Containing Analyte Any sample which is reasonablysuspected of containing analyte can be analyzed by the methods of thepresent disclosure. Such samples can include human, animal or man-madesamples. The sample can be prepared in any convenient medium which doesnot interfere with the assay. Typically, the sample is an aqueoussolution or a natural fluid, such as, but not limited to, urine, wholeblood, serum, plasma, cerebral-spinal fluid, or saliva. In someinstances, the sample is serum.

Measuring the Amount of Analyte

Quantitative, semiquantitative, and qualitative methods as well as allother methods for determining analyte are considered to be methods ofmeasuring the amount of analyte. For example, a method which merelydetects the presence or absence of analyte in a sample suspected ofcontaining an analyte is considered to be included within the scope ofthe present disclosure.

Synonyms for the phrase “measuring the amount of analyte” which arecontemplated within the scope of the present disclosure include, but arenot limited to, detecting, measuring, or determining analyte; detecting,measuring, or determining the presence of analyte; detecting, ordetermining the amount of analyte; and detecting, measuring ordetermining the concentration of analyte.

Member of a Specific Binding Pair

A member of a specific binding pair (sbp member) is one of two differentmolecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand), sbp member and sbp partner, or the like. Thesewill usually be members of an immunological pair such asantigen-antibody.

Ligand

Any organic compound for which a receptor naturally exists or can beprepared. For example, in one context of the present disclosure, theanalyte is a ligand and the present disclosure provides methods fordetermining the amount or concentration of the analyte which is aligand.

Receptor

A receptor is any compound or composition capable of recognizing aparticular spatial and polar organization of a molecule. These organizedareas of a molecule are referred to as epitopic or determinant sites.Illustrative naturally occurring receptors include antibodies andenzymes.

Epitope

“Epitope” is a molecular region on the surface of an antigen capable ofeliciting an immune response and of combining with the specific antibodyproduced by such a response, also called determinant, antigenicdeterminant. In reference to a hapten (such as symmetricdimethylarginine) an antibody can be generated against the non-antigenichapten molecule by conjugating the hapten to an immunogenic carrier. Anantibody is then generated which recognizes an “epitope” defined by thehapten.

Linking Group

A linking group is a portion of a structure which connects two or moresubstructures. A linking group has at least one uninterrupted chain ofatoms extending between the substructures. The atoms of a linking groupare themselves connected by chemical bonds. The number of atoms in alinking group is determined by counting the atoms other than hydrogen.

Conjugate

A conjugate is a molecule comprised of two or more substructures boundtogether through a linking group to form a single structure. The bindingcan be made by connecting the subunits through a linking group. Withinthe context of the present disclosure, a conjugate can include aglucose-6-phosphate dehydrogenase (G6PDH) enzyme attached to a hapten,sbp member or analyte analog, such as a conjugate where a G6PDH mutantenzyme is used (e.g., recombinant G6PDH as described in U.S. Pat. Nos.6,455,288, 6,090,567, and 6,033,890). Within the context of the presentdisclosure, G6PDH may also be referred to as an enzyme, such as a G6PDHenzyme, or a label, such as a G6PDH label. In some cases, a conjugatecan include a label (e.g., a label protein) including, but not limitedto, G6PDH, alkaline phosphatase, β-galactosidase, and horse radishperoxidase, or a chemical label such as a fluorescent, luminescent orcolorimetric molecule attached to a hapten, sbp member or analyteanalog.

Conjugation

Conjugation is any process where two subunits are linked together toform a conjugate. The conjugation process can be comprised of any numberof steps, for example as described herein.

Hapten

Haptens are capable of binding specifically to corresponding antibodies,but usually do not themselves act as immunogens for preparation of theantibodies. Antibodies which recognize a hapten can be prepared againstcompounds comprised of the hapten linked to an immunogenic carrier.

Symmetrically Dimethylated Arginine Analyte

“Symmetrically dimethylated arginine analyte” refers to analytes havingan antibody-binding epitope which is common to symmetricdimethylarginine. Analytes included in symmetrically dimethylatedarginine analytes include symmetric dimethylarginine (SDMA), symmetricNα-acylated-dimethylarginine, such as symmetricNα-acetyl-dimethylarginine, and SDMA-peptide derivatives, such asSDMA-Gly-Gly dipeptide.

Derivative

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

Analog

The term “analog” is a compound having a structure similar to that ofanother compound, but differing from it in respect to a certaincomponent. It can differ in one or more atoms, functional groups, orsubstructures, which are replaced with other atoms, groups, orsubstructures.

Label

A “label,” “detector molecule,” “reporter” or “detectable marker” is anymolecule which produces, or can be induced to produce, a detectablesignal. The label can be conjugated to an analyte, immunogen, antibody,or to another molecule such as a receptor or a molecule that can bind toa receptor such as a ligand, particularly a hapten or antibody. A labelcan be attached directly or indirectly by a linking group. Non-limitingexamples of labels include radioactive isotopes (e.g., ¹²⁵I), enzymes(e.g. β-galactosidase, peroxidase), G6PDH (e.g., mutant G6PDH, such asrecombinant G6PDH as described in U.S. Pat. Nos. 6,455,288, 6,090,567,and 6,033,890), enzyme fragments, enzyme substrates, enzyme inhibitors,coenzymes, catalysts, fluorophores (e.g., rhodamine, fluoresceinisothiocyanate or FITC, or Dylight 649), dyes, chemiluminescers andluminescers (e.g., dioxetanes, luciferin), or sensitizers.

Immunogen

The term “immunogen” refers to a substance capable of eliciting,producing, or generating an immune response in an organism.

Immunogenic Carrier

An “immunogenic carrier,” as used herein, is an immunogenic substance,commonly a protein, that can join at one or more positions with haptens,thereby enabling the production of antibodies that can specifically bindwith these haptens. Examples of immunogenic carrier substances include,but are not limited to, proteins, glycoproteins, complexpolyamino-polysaccharides, particles, and nucleic acids that arerecognized as foreign and thereby elicit an immunologic response fromthe host. The polyamino-polysaccharides may be prepared frompolysaccharides using any of the conventional means known for thispreparation.

Protein

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein to refer to a polymeric form of amino acids ofany length. Unless specifically indicated otherwise, “polypeptide,”“peptide,” and “protein” can include genetically coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, polypeptides having modified peptide backbones, and fusionproteins.

Signal Producing System

The “signal producing system” is utilized in assays for analytes and mayhave one or more components, at least one component being a detectablelabel (e.g., G6PDH, such as a mutant G6PDH). The signal producing systemgenerates a signal that relates to the presence or amount of analyte ina sample. The signal producing system includes all of the reagentsrequired to produce a measurable signal. For purposes of the presentdisclosure, typically, the G6PDH or a label protein (e.g., alkalinephosphatase, β-galactosidase or horse radish peroxidase) is conjugatedto a sbp member analogous to the analyte.

Other components of the signal producing system can include substrates,enhancers, activators, chemiluminescent compounds, cofactors,inhibitors, scavengers, metal ions, specific binding substances requiredfor binding of signal generating substances, coenzymes, substances thatreact with enzymic products, other enzymes and catalysts, and the like.

The signal producing system provides a signal detectable by externalmeans, such as by measurement of electromagnetic radiation, e.g., byvisual examination. In some instances, the signal producing systemincludes a chromophoric substrate and an enzyme label (e.g., mutantG6PDH enzyme), where chromophoric substrates are enzymatically convertedto dyes which absorb light in the ultraviolet or visible region.

Isolated

“Isolated” when used in the context of an antibody means altered “by thehand of man” from any natural state; i.e., that, if it occurs in nature,it has been changed or removed from its original environment, or both.For example, a naturally occurring antibody naturally present in aliving animal in its natural state is not “isolated”, but the sameantibody separated from the coexisting materials of its natural state is“isolated”, as the term is employed herein. Antibodies may occur in acomposition, such as an immunoassay reagent, which are not naturallyoccurring compositions, and therein remain isolated antibodies withinthe meaning of that term as it is employed herein.

Cross-Reactivity

“Cross-reactivity” refers to the reaction of an antibody with an antigenthat was not used to induce that antibody. “Cross-reactivity” may bedetermined in a quantitative immunoassay by establishing a standardcurve using known dilutions of the target analyte. The standard curve isthen used to calculate the apparent concentration of the interferingsubstance present in various known amounts in samples assayed undersimilar condition. The cross-reactivity is the apparent concentrationdivided by the actual concentration multiplied by 100.

Calibration and Control Material

The phrase “calibration and control material” refers to any standard orreference material containing a known amount of an analyte. A samplesuspected of containing an analyte and the corresponding calibrationmaterial are assayed under similar conditions. The concentration ofanalyte is calculated by comparing the results obtained for the unknownspecimen or sample containing known concentration of analyte with theresults obtained for the standard. This is commonly done by constructingor generating a calibration curve.

Sensitivity

Is used in the sense of detection limit, i.e., the smallest amount of ananalyte giving a signal that is distinguishable from the signal obtainedin the absence of analyte.

Spike-Recovery

“Spike-recovery” refers to an assay measuring the amount of analyte(recovery) in a sample mixture compared to a known amount of the analyteadded (spiked) to the sample mixture. The measuring the amount ofanalyte may be expressed in terms of concentration (ng/mL) or apercentage (%).

Substantial Change in Enzyme Activity

A change in activity of an enzyme sufficient to allow detection of ananalyte when the enzyme is used as a label in an assay for the analyte.Typically, the enzyme's activity is reduced 10 to 100%, such as 20 to99%, or 30 to 95%.

Inhibitory Antibody

An antibody capable of inhibiting the activity of an enzyme or anenzyme-ligand conjugate upon binding an epitope present on the enzyme.Such antibodies are distinguished from anti-ligand antibodies capable ofinhibiting the enzyme activity of enzyme-ligand conjugates upon bindingto the ligand.

Modulation

In an assay experiment “modulation” refers to hapten or analyte attachedto a label such as an enzyme and an analyte in a sample suspected ofcontaining the analyte competing for analyte-antibody binding sites,thus modulating the amount of enzymatic product formed (see FIG. 10).

Maximum Inhibition

“Maximum inhibition” refers to an antibody capable of inhibiting theactivity of an enzyme or an enzyme-ligand conjugate upon binding anepitope present on the enzyme when excess antibody is added to the assayand the signal obtained in the absence of analyte.

Ancillary Materials

Various ancillary materials will frequently be employed in an assay inaccordance with the present disclosure. For example, buffers willnormally be present in the assay medium, as well as stabilizers for theassay medium and the assay components. Frequently, in addition to theseadditives, additional proteins may be included, such as albumins, orsurfactants, particularly non-ionic surfactants, binding enhancers,e.g., polyalkylene glycols, or the like.

Compounds, Conjugates and Syntheses Thereof

Homogeneous enzyme immunoassays depend on the availability of enzyme-sbpmember conjugates whose enzyme activity can be strongly modulated onbinding of the sbp partner. The present disclosure provides enzyme-sbpmember conjugates and antibodies for conducting assays that are usefulin homogeneous immunoassays.

In certain embodiments, protein immunogens are synthesized and used toprepare antibodies specific for compounds, such as a symmeticallydimethylated arginine analyte. The antibodies may be used in methods fordetecting a symmetically dimethylated arginine analyte in a samplesuspected of containing the analyte. Label conjugates are prepared andmay be employed in the above methods. Effective screening of samples forthe presence of one or more symmetrically dimethylated arginine analytesas referred to above may be realized.

The immunogens and label conjugates may involve a derivative ofsymmetric dimethylarginine linked through the nitrogen atom of theα-amino group of symmetric dimethylarginine to a protein or a label. Insome instances, the conjugate may be referred to herein as a proteinconjugate or a label conjugate, respectively.

Compounds of the present disclosure include compounds useful forproducing antibodies according to the present disclosure. In addition,compounds of the present disclosure include conjugates useful for theimmunoassays described herein. In certain embodiments, the compoundsinclude a compound of Formula 1:

wherein:

R¹ is —Y—Z;

Y is a linking group; and

Z is selected from the group consisting of hydrogen, OH, SH, S-acyl,O-alkyl, halogen, NH₂, epoxy, maleimidyl, haloacetamide, carboxyl,activated carboxyl, an immunogenic carrier, a protein, and a label.

In some embodiments, Z is a protein. For example, the protein can be animmunogenic carrier. The immunogenic carrier can be conjugated to asymmetric dimethylarginine hapten, thereby enabling the production ofantibodies that can specifically bind with the hapten. For example, theimmunogenic carrier can be selected from a hemocyanin, a globulin, analbumin, and a polysaccharide. In some instances, the immunogeniccarrier is bovine serum albumin (BSA). In some instances, theimmunogenic carrier is keyhole limpet hemocyanin (KLH). In certainembodiments, the immunogenic carrier may be modified to include one ormore functional groups. The functional group on the modified immunogeniccarrier can be a reactive functional group that facilitates attachmentof the immunogenic carrier to the linking group in the compound ofFormula 1.

In some embodiments, a symmetric dimethylarginine hapten is alkylated onthe nitrogen atom of the α-amino group of symmetric dimethylarginine(FIG. 1). As such, in some cases, the linking group comprises an alkylor substituted alkyl group attached to the nitrogen atom of the α-aminogroup of symmetric dimethylarginine (i.e., attached to the R¹-nitrogenatom). In certain embodiments, such haptens are used to produceantibodies specific for symmetrically dimethylated arginine analytes.

In certain embodiments, Z is a label. The label is a molecule whichproduces, or can be induced to produce, a detectable signal. Forexample, the label can be an enzyme, such as an enzyme selected from analkaline phosphatase, a β-galactosidase and a horse radish peroxidase.In some embodiments, the label is an enzyme, where the enzyme isglucose-6-phosphate dehydrogenase (G6PDH). In some instances, the G6PDHis a mutant G6PDH, which includes one or more amino acid residuesubstitutions relative to the wild-type form. For example, the mutantG6PDH can include a cysteine substitution, e.g., a cysteine substitutionin each subunit of the G6PDH enzyme. In some cases, the linking groupcan be attached to the G6PDH enzyme at the cysteine residue. In certainembodiments, the label may be modified to include one or more functionalgroups. The functional group on the modified label can be a reactivefunctional group that facilitates attachment of the label to the linkinggroup in the compound of Formula 1.

In some embodiments, a symmetric dimethylarginine derivative is acylatedon the nitrogen atom of the α-amino group of symmetric dimethylarginine(FIG. 1). As such, in some cases, the linking group comprises an acyl orsubstituted acyl group attached to the nitrogen atom of the α-aminogroup of symmetric dimethylarginine (i.e., attached to the R¹-nitrogenatom). In certain embodiments, such derivatives are used to produceconjugates useful in the immunoassays described herein.

In certain embodiments, Z is a protein. The protein can be anyconvenient protein, which includes amino acid residues, such as adipeptide, tripeptide, and the like, in any number of such amino acidresidues, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more,7 or more, 8 or more, 9 or more, 10 or more, etc. In certainembodiments, the protein may be modified to include one or morefunctional groups. The functional group on the modified protein can be areactive functional group that facilitates attachment of the protein tothe linking group in the compound of Formula 1. In some instances, theprotein is acylated. In some instances, the protein is alkylated.

The linking group may include about 1 to 25 atoms (excluding hydrogenatoms) and may include a chain of from 2 to 15 atoms (excluding hydrogenatoms), each independently selected from carbon, oxygen, sulfur,nitrogen, halogen and phosphorous. In some embodiments, the linkinggroup includes 1 to 15 carbon atoms and/or 0 to 6 heteroatoms. Examplesof linking groups include, but are not limited to, —(CH₂)_(n)C(O)—,—C(O)(CH₂)_(n)—, —C(O)(CH₂)_(n)NHC(O)—, —C(O)(CH₂)_(n)NHC(O)(CH₂)_(n)—,—(CH₂)_(n)SCH₂C(O)—, —(CH₂)_(n)C(O)NH(CH₂)_(n)—, —(CH₂)_(n)NHC(O)—,—(CH₂)_(n)NHC(O)(CH₂)_(n)—, —NH(CH₂)C(O)—, —(CH₂)_(n)—, and—(CH₂)_(n)(heterocyclyl)S(CH₂)_(n)C(O)—, and n is an integer from 1 to10, and including acid salts thereof. In certain embodiments, thelinking group is —C(O)(CH₂)_(n)NHC(O)(CH₂)_(n)—, such as—C(O)(CH₂CH₂)NHC(O)(CH₂)—. In certain embodiments, the linking group is—(CH₂)_(n)(heterocyclyl)S(CH₂)_(n)C(O)—, such as—(CH₂CH₂CH₂CH₂)(2,5-dioxopyrrolidin-1-yl)S(CH₂)C(O)—.

The number of heteroatoms in the linking group may range from 0 to 6,such as from about 1 to 5, or from 2 to 5, or from 3 to 5. The linkingagents may be aliphatic or aromatic. When heteroatoms are present,oxygen may be present as oxo or oxy, bonded to carbon, sulfur, nitrogenor phosphorous; nitrogen may be present as nitro, nitroso or amino,bonded to carbon, oxygen, sulfur or phosphorous; sulfur can be analogousto oxygen; phosphorous can be bonded to carbon, sulfur, oxygen ornitrogen, such as phosphonate and phosphate mono or di-ester. Commonfunctionalities in forming a covalent bond between the linking group andthe molecule to be conjugated are alkylamine, amidine, thioamide, ether,urea, thiourea, guanidine, azo, thioether and carboxylate, sulfonate,and phosphate esters, amides and thioesters.

In certain embodiments, when a linking group has a non-oxocarbonyl groupincluding nitrogen and sulfur analogs, a phosphate group, an aminogroup, alkylating agent such as halo or tosylalkyl, oxy (hydroxyl or thesulfur analog, mercapto) oxocarbonyl (e.g., aldehyde or ketone), oractive olefin such as a vinyl sulfone or α-, β-unsaturated ester, thesefunctionalities can be linked to amine groups, carboxyl groups, activeolefins, alkylating agents, e.g., bromoacetyl. Where an amine andcarboxylic acid or its nitrogen derivative or phosphoric acid is linked,amides, amidines and phosphoramides can be formed. Where mercaptan andactivated olefin are linked, thioethers can be formed. Where a mercaptanand an alkylating agent are linked, thioethers can be formed. Wherealdehyde and an amine are linked under reducing conditions, analkylamine can be formed. Where a carboxylic acid or phosphate acid andan alcohol are linked, esters can be formed. Various linking groups aredescribed, see, for example, Cautrecasas, J. Biol. Chem. (1970)245:3059.

To develop an assay for symmetric dimethylarginine, the chemicalstructure of symmetric dimethylated arginine analytes is used. Forexample, asymmetric dimethylarginine has two methyl groups added to oneof the terminal nitrogen atoms of the guanidium group (see FIG. 2).Monomethyl arginine has a single methyl group on one of the terminalnitrogen atoms (FIG. 2). Symmetric dimethylarginine has two methylgroups, one methyl group added to each of the terminal nitrogen atoms ofthe guanidine group (FIG. 1). The specific symmetrical methylation ofthe chemical structures are retained to prepare immunogens and raiseantibodies accordingly.

The present disclosure provides for the design of symmetricdimethylarginine haptens and immunogens by modification of the nitrogenatom of the α-amino group of symmetric dimethylarginine. Haptens SDMA-Mand SDMA-SBAP of the present disclosure are shown in FIG. 4. Placementof a linking group at the amine nitrogen of symmetric dimethylarginineprovides for antibodies that may specifically react with a symmetricallydimethylated arginine analyte because the analytes share the symmetricdimethylated guanidine group. The present disclosure thus providessymmetric dimethylarginine derivatives (i.e., symmetrically dimethylatedarginine analytes) and immunogens useful with the various types ofimmunoassays described herein (see, e.g., FIG. 10).

Compounds useful for producing antibodies and conjugates according tothe present disclosure can be synthesized in accordance with the generalsynthetic methods described below. Compounds of Formula 1 can beprepared by standard methods. The following reaction schemes are onlymeant to represent examples of the methods and are in no way meant tolimit the present disclosure.

a) Haptens

Attachment of maleimide functionality to the nitrogen atom of theα-amino group of symmetric dimethylarginine may be accomplished throughuse of 4-maleimido-1-butanal (4) shown in Scheme 1, the preparation ofwhich is described in Example 1. For the alkylation reaction on thenitrogen atom of the α-amino group of symmetric dimethylarginine, AcOHand NaBH₃CN can be added to a solution of 4-maleimido-1-butanal (4) andsymmetric dimethylarginine in MeOH. The resulting mixture can be stirredat room temperature for 3 hours. The reaction can be quenched with waterand purified by reverse phase chromatography to give the hapten SDMA-M(FIG. 4).

Attachment of a linking group to the nitrogen atom of the α-amino groupof symmetric dimethylarginine may be accomplished through use ofN-tert-(butoxycarbonyl)-β-alanine (6) shown in Scheme 2, the preparationof which is described in Example 2. Acylation of symmetricaldimethylarginine with N-tert-(butoxycarbonyl)-β-alanine (6) is alsodescribed in Example 2. Deprotection as described in Example 2 mayprovide the compound (8) which may be further elaborated by reactingwith N-succinimidyl bromoacetate (9) and DIPEA in DMF. Solvent can beremoved under vacuum. The crude product can be dissolved in MeCN andwater and then purified with reverse phase chromatography to give theproduct SDMA-SBAP (FIG. 4). The resulting product (SDMA-SBAP) can belinked to thiol containing proteins.

Removal of the t-Boc protecting group from the linking group can producethe compound (8). Suitable protecting groups are described in detail inpatents and articles in the technical literature. See, for example,“Principles of Peptide Synthesis” (M. Bodanszky, Springer Verlag,Berlin, Heidelberg, New York, Tokyo (1984)). Examples of such protectinggroups, by way of example and not limitation, are t-butoxycarbonyl(t-Boc), fluorenylmethyloxycarbonyl (Fmoc), acetaminomethyl (Acm),triphenyl methyl (Trt), benzyloxycarbonyl, biphenylisopropyloxycarbonyl,1-amyloxycarbonyl, isobornyloxycarbonyl,alpha-dimethyl-3,5-dimethoxybenxyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-1,1-dimentylethoxycarbonyl, bromobenzyloxy, carbamyl, formyl,and the like. The particular protecting group chosen may depend on thenature of the reaction to be performed and the conditions of suchreaction such as temperature, pH, and so forth.

b) Immunogen

Maleimide functionalized haptens (e.g. SDMA-M) may be conjugated toproteins. Activation of protein lysine residues by acylation of theepsilon-nitrogen with N-succinimidyl S-acetylthioacetate (SATA),followed by subsequent hydrolysis of the S-acetyl group withhydroxylamine produces a nucleophilic sulfhydryl group. Conjugation ofthe sulfhydryl activated protein with the maleimide derivatized haptenproceeds via a Michael addition reaction. Suitable proteins (immunogeniccarriers) include, but are not limited to, keyhole limpet hemocyanin,bovine thyroglobulin, and ovalbumin.

Compound SDMA-M includes the maleimide functionality for thiolmodification of thiol containing proteins. The synthesis of symmetricdimethylarginine immunogen SDMA-M-SH-KLH with a linking group on thenitrogen atom of the α-amino group of symmetric dimethylarginine beginswith the synthesis of SDMA-M as shown in FIG. 5, the preparation ofwhich is described in Example 1. Reaction of amines from keyhole limpethemocyanin (KLH) with N-succinimidyl S-acetylthioacetate can produceprotected sulfhydryls that can be subsequently deprotected byhydroxylamine for reaction with SDMA-M. Reaction of thiol modified KLHwith SDMA-M in sodium phosphate (0.1 M, pH=8.0) buffer solution canproduce the desired immunogen SDMA-M-SH-KLH as show in FIG. 8. Theimmunogen SDMA-M-SH-KLH can be purified by chromatography, such as on aSephadex G-25 column with buffer solution. The concentration ofimmunogen SDMA-M-SH-KLH can be measured using a protein assay, such as,but not limited to a Pierce™ Rapid Gold BCA protein assay kit. Theimmunogen SDMA-M-SH-KLH can be used for the immunization of rabbits forantibody production.

c) Enzyme Conjugate

Hapten SDMA-SBAP with bromoacetamide functionality can be used forreaction with proteins containing a thiol group. Conjugation ofSDMA-SBAP to cysteine containing G6PDH is shown in FIG. 7, thepreparation of which is described in Example 2.

Hapten SDMA-M can be used to prepare immunogen. Hapten SDMA-SBAP can beused to prepare a G6PDH conjugate. The immunogen SDMA-M-SH-KLH can beused for elicitation of antibodies. In certain embodiments, in anenzyme-based assay format, antibodies produced can show good modulationwith a symmetrically dimethylated arginine analyte. In some embodiments,the immunogen SDMA-M-SH-KLH can be used to successfully raiseantibodies, which may provide an indication that such antibodies havepotential use in an enzyme-based symmetric dimethylarginine immunoassayas described hereinafter.

Antibodies and Preparation Thereof

Aspects of the present disclosure include antibodies, which specificallybind to symmetrically dimethylated arginine analytes. In some instances,that antibodies specifically bind one or more of asymmetricdimethylarginine, L-arginine, and N-methylarginine. In some embodiments,an antibody of the present disclosure specifically binds to any of thecompounds of the present disclosure, including any of the compounds ofFormula 1 described elsewhere herein.

The term “antibody” (also used interchangeably with “immunoglobulin”)encompasses polyclonal (e.g., rabbit polyclonal) and monoclonal antibodypreparations where the antibody may be an antibody or immunoglobulin ofany isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA,IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramerwhich in turn is composed of two dimers of a heavy and light chainpolypeptide); single chain antibodies (e.g., scFv); fragments ofantibodies (e.g., fragments of whole or single chain antibodies) whichretain specific binding to the compound, including, but not limited tosingle chain Fv (scFv), Fab, (Fab′)₂, (scFv′)₂, and diabodies; chimericantibodies; monoclonal antibodies, human antibodies; and fusion proteinscomprising an antigen-binding portion of an antibody and a non-antibodyprotein. In some embodiments, the antibody is selected from an IgG, Fv,single chain antibody, scFv, Fab, F(ab′)2, or Fab′. The antibodies maybe further conjugated to other moieties, such as members of specificbinding pairs, e.g., biotin (member of biotin-avidin specific bindingpair), and the like.

Immunoglobulin polypeptides include the kappa and lambda light chainsand the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta, epsilon and muheavy chains or equivalents in other species. Full-length immunoglobulin“light chains” (usually of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 150 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

An immunoglobulin light or heavy chain variable region is composed of a“framework” region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs have been defined (see, “Sequences ofProteins of Immunological Interest,” E. Kabat et al., U.S. Department ofHealth and Human Services, (1991 and Lefranc et al. IMGT, theinternational ImMunoGeneTics information System®. Nucl. Acids Res.,2005, 33, D593-D597)). A detailed discussion of the IMGT system,including how the IMGT system was formulated and how it compares toother systems, is provided on the World Wide Web atimgt.cines.fr/textes/IMGTScientificChart/Numbering/IMGTnumberingsTable.html.The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs. The CDRs are primarily responsible for binding to an epitope of anantigen. All CDRs and framework provided by the present disclosure aredefined according to IMGT, supra, unless otherwise indicated.

An “antibody” thus encompasses a protein having one or more polypeptidesthat can be genetically encodable, e.g., by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies encompass intact immunoglobulins as well as a number of wellcharacterized fragments which may be genetically encoded or produced bydigestion with various peptidases. Thus, for example, pepsin digests anantibody below the disulfide linkages in the hinge region to produceF(ab)′₂, a dimer of Fab which itself is a light chain joined to VH-CHIby a disulfide bond. The F(ab)′₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region thereby converting the(Fab′)₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially aFab with part of the hinge region (see, Fundamental Immunology, W. E.Paul, ed., Raven Press, N.Y. (1993), for a more detailed description ofother antibody fragments). While various antibody fragments are definedin terms of the digestion of an intact antibody, one of skill willappreciate that such Fab′ fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein also includes antibody fragments eitherproduced by the modification of whole antibodies or synthesized de novousing recombinant DNA methodologies, including, but are not limited to,Fab′₂, IgG, IgM, IgA, scFv, dAb, nanobodies, unibodies, and diabodies.In certain embodiments, an antibody of the present disclosure isselected from an IgG, Fv, single chain antibody, scFv, Fab, F(ab′)₂, andFab′.

The phrases “specifically binds”, “specific for”, “immunoreactive” and“immunoreactivity”, and “antigen binding specificity”, when referring toan antibody, refer to a binding reaction with an antigen which is highlypreferential to the antigen or a fragment thereof, so as to bedeterminative of the presence of the antigen in the presence of aheterogeneous population of antigens. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular antigen and donot bind in a significant amount to other antigens present in thesample. Specific binding to an antigen under such conditions may requirean antibody that is selected for its specificity for a particularantigen. For example, the antibodies may specifically bind to thecompound, and do not exhibit comparable binding to other moleculespresent in a sample.

In some embodiments, an antibody of the present disclosure “specificallybinds” to the compound if it binds to or associates with the compoundwith an affinity or K_(a) (that is, an equilibrium association constantof a particular binding interaction with units of 1/M) of, for example,greater than or equal to about 10⁵ M⁻¹. In certain embodiments, theantibody binds to the compound with a K_(a) greater than or equal toabout 10⁶ M⁻¹, 10⁷ M⁻¹, 108 M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10 M⁻¹, 10¹² M⁻¹, or10¹³ M⁻¹. “High affinity” binding refers to binding with a K_(a) of atleast 10⁷ M⁻¹, at least 108 M⁻¹, at least 10⁹ M⁻¹, at least 10¹⁰ M⁻¹, atleast 10¹¹ M⁻¹, at least 10¹² M⁻¹, at least 10¹³ M⁻¹, or greater.Alternatively, affinity may be defined as an equilibrium dissociationconstant (K_(D)) of a particular binding interaction with units of M(e.g., 10-5 M to 10⁻¹³ M, or less). In some embodiments, specificbinding means the antibody binds to the compound with a K_(D) of lessthan or equal to about 10-5 M, less than or equal to about 10-6 M, lessthan or equal to about 10-7 M, less than or equal to about 10-8 M, orless than or equal to about 10-9 M, 10-1⁰ M, 10⁻¹¹ M, or 10⁻¹² M orless. The binding affinity of the antibody for the compound can bereadily determined using conventional techniques, e.g., by competitiveELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, byusing surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000instrument, using general procedures outlined by the manufacturer); byradioimmunoassay; or the like.

Whether a first antibody “competes with” a second antibody for bindingto the compound may be readily determined using competitive bindingassays known in the art. Competing antibodies may be identified, forexample, via an antibody competition assay. For example, a sample of afirst antibody can be bound to a solid support. Then, a sample of asecond antibody suspected of being able to compete with such firstantibody is then added. One of the two antibodies is labelled. If thelabeled antibody and the unlabeled antibody bind to separate anddiscrete sites on the compound, the labeled antibody will bind to thesame level whether or not the suspected competing antibody is present.However, if the sites of interaction are identical or overlapping, theunlabeled antibody will compete, and the amount of labeled antibodybound to the compound will be lowered. If the unlabeled antibody ispresent in excess, very little, if any, labeled antibody will bind.

For purposes of the present disclosure, competing antibodies are thosethat decrease the binding of an antibody to the compound by about 50% ormore, about 60% or more, about 70% or more, about 80% or more, about 85%or more, about 90% or more, about 95% or more, or about 99% or more.Details of procedures for carrying out such competition assays are wellknown in the art and can be found, for example, in Harlow and Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988, 567-569, 1988, ISBN 0-87969-314-2. Suchassays can be made quantitative by using purified antibodies. A standardcurve may be established by titrating one antibody against itself, i.e.,the same antibody is used for both the label and the competitor. Thecapacity of an unlabeled competing antibody to inhibit the binding ofthe labeled antibody to the plate may be titrated. The results may beplotted, and the concentrations necessary to achieve the desired degreeof binding inhibition may be compared.

According to some embodiments, an antibody of the present disclosurecompetes for binding to the compound of Formula 1 with an antibodycomprising:

a variable heavy chain (V_(H)) polypeptide comprising

-   -   a V_(H) CDR1 comprising the amino acid sequence GFSLSSY (SEQ ID        NO:2),    -   a V_(H) CDR2 comprising the amino acid sequence DIKTGDR (SEQ ID        NO:3), and    -   a V_(H) CDR3 comprising the amino acid sequence ARVYVSGNDHYDL        (SEQ ID NO:4); and

a variable light chain (V_(L)) polypeptide comprising

-   -   a V_(L) CDR1 comprising the amino acid sequence QSISNY (SEQ ID        NO:6),    -   a V_(L) CDR2 comprising the amino acid sequence RAS (SEQ ID        NO:7), and    -   a V_(L) CDR3 comprising the amino acid sequence QLGYTYSNVENA        (SEQ ID NO:8).

In certain embodiments, such an antibody comprises the six CDRs setforth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, and SEQ ID NO:8. According to some embodiments, the antibodycomprises: a variable heavy chain (V_(H)) polypeptide comprising anamino acid sequence having 70% or greater, 75% or greater, 80% orgreater, 85% or greater, 90% or greater, or 95% or greater identity tothe amino acid sequence set forth in SEQ ID NO:1; and a variable lightchain (V_(L)) polypeptide comprising an amino acid sequence having 70%or greater, 75% or greater, 80% or greater, 85% or greater, 90% orgreater, or 95% or greater identity to the amino acid sequence set forthin SEQ ID NO:5.

According to some embodiments, an antibody of the present disclosurecompetes for binding to the compound of Formula 1 with an antibodycomprising:

a variable heavy chain (V_(H)) polypeptide comprising

-   -   a V_(H) CDR1 comprising the amino acid sequence GFSLSSY (SEQ ID        NO:2),    -   a V_(H) CDR2 comprising the amino acid sequence DIKTGDR (SEQ ID        NO:3), and    -   a V_(H) CDR3 comprising the amino acid sequence ARVYVSGNDHYDL        (SEQ ID NO:4); and

a variable light chain (V_(L)) polypeptide comprising

-   -   a V_(L) CDR1 comprising the amino acid sequence QSISNY (SEQ ID        NO:6),    -   a V_(L) CDR2 comprising the amino acid sequence RAS (SEQ ID        NO:7), and    -   a V_(L) CDR3 comprising the amino acid sequence QLGYTYTNVENA        (SEQ ID NO:10).

In certain embodiments, such an antibody comprises the six CDRs setforth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, and SEQ ID NO:10. According to some embodiments, the antibodycomprises: a variable heavy chain (V_(H)) polypeptide comprising anamino acid sequence having 70% or greater, 75% or greater, 80% orgreater, 85% or greater, 90% or greater, or 95% or greater identity tothe amino acid sequence set forth in SEQ ID NO:1; and a variable lightchain (V_(L)) polypeptide comprising an amino acid sequence having 70%or greater, 75% or greater, 80% or greater, 85% or greater, 90% orgreater, or 95% or greater identity to the amino acid sequence set forthin SEQ ID NO:9.

According to some embodiments, an antibody of the present disclosurecompetes for binding to the compound of Formula 1 with an antibodycomprising:

a variable heavy chain (V_(H)) polypeptide comprising

-   -   a V_(H) CDR1 comprising the amino acid sequence GFSFSSTK (SEQ ID        NO:12),    -   a V_(H) CDR2 comprising the amino acid sequence CIGTDT (SEQ ID        NO:13), and    -   a V_(H) CDR3 comprising the amino acid sequence ARSSSTGYYNL (SEQ        ID NO:14); and

a variable light chain (V_(L)) polypeptide comprising

-   -   a V_(L) CDR1 comprising the amino acid sequence QSIRSY (SEQ ID        NO:16),    -   a V_(L) CDR2 comprising the amino acid sequence YAS (SEQ ID        NO:17), and    -   a V_(L) CDR3 comprising the amino acid sequence HDYYTFTDND (SEQ        ID NO:18).

In certain embodiments, such an antibody comprises the six CDRs setforth in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:17, and SEQ ID NO:18. According to some embodiments, the antibodycomprises: a variable heavy chain (V_(H)) polypeptide comprising anamino acid sequence having 70% or greater, 75% or greater, 80% orgreater, 85% or greater, 90% or greater, or 95% or greater identity tothe amino acid sequence set forth in SEQ ID NO:11; and a variable lightchain (V_(L)) polypeptide comprising an amino acid sequence having 70%or greater, 75% or greater, 80% or greater, 85% or greater, 90% orgreater, or 95% or greater identity to the amino acid sequence set forthin SEQ ID NO:15.

According to some embodiments, an antibody of the present disclosurecompetes for binding to the compound of Formula 1 with an antibodycomprising:

a variable heavy chain (V_(H)) polypeptide comprising

-   -   a V_(H) CDR1 comprising the amino acid sequence GFSFSSTK (SEQ ID        NO:12);    -   a V_(H) CDR2 comprising the amino acid sequence CIGVGSRGS (SEQ        ID NO:20); and    -   a V_(H) CDR3 comprising the amino acid sequence ARSSTTGYYIL (SEQ        ID NO:21); and

a variable light chain (V_(L)) polypeptide comprising

-   -   a V_(L) CDR1 comprising the amino acid sequence ESIYSY (SEQ ID        NO:23);    -   a V_(L) CDR2 comprising the amino acid sequence KAS (SEQ ID        NO:24); and    -   a V_(L) CDR3 comprising the amino acid sequence QNYYTFTEND (SEQ        ID NO:25).

In certain embodiments, such an antibody comprises the six CDRs setforth in SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:24, and SEQ ID NO:25. According to some embodiments, the antibodycomprises: a variable heavy chain (V_(H)) polypeptide comprising anamino acid sequence having 70% or greater, 75% or greater, 80% orgreater, 85% or greater, 90% or greater, or 95% or greater identity tothe amino acid sequence set forth in SEQ ID NO:19; and a variable lightchain (V_(L)) polypeptide comprising an amino acid sequence having 70%or greater, 75% or greater, 80% or greater, 85% or greater, 90% orgreater, or 95% or greater identity to the amino acid sequence set forthin SEQ ID NO:22.

According to some embodiments, an antibody of the present disclosurecompetes for binding to the compound of Formula 1 with an antibodycomprising:

a variable heavy chain (V_(H)) polypeptide comprising

-   -   a V_(H) CDR1 comprising the amino acid sequence GFSFWR (SEQ ID        NO:27);    -   a V_(H) CDR2 comprising the amino acid sequence CIDGGNTNR (SEQ        ID NO:28); and    -   a V_(H) CDR3 comprising the amino acid sequence ARVRLGNNDYIDL        (SEQ ID NO:29); and

a variable light chain (V_(L)) polypeptide comprising

-   -   a V_(L) CDR1 comprising the amino acid sequence QSISNY (SEQ ID        NO:6);    -   a V_(L) CDR2 comprising the amino acid sequence RAS (SEQ ID        NO:7); and    -   a V_(L) CDR3 comprising the amino acid sequence QQGYNWDLDGA (SEQ        ID NO:31).

In certain embodiments, such an antibody comprises the six CDRs setforth in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:6, SEQ IDNO:7, and SEQ ID NO:31. According to some embodiments, the antibodycomprises: a variable heavy chain (V_(H)) polypeptide comprising anamino acid sequence having 70% or greater, 75% or greater, 80% orgreater, 85% or greater, 90% or greater, or 95% or greater identity tothe amino acid sequence set forth in SEQ ID NO:26; and a variable lightchain (V_(L)) polypeptide comprising an amino acid sequence having 70%or greater, 75% or greater, 80% or greater, 85% or greater, 90% orgreater, or 95% or greater identity to the amino acid sequence set forthin SEQ ID NO:30.

The amino acid sequences of the above-referenced variable heavy chain(V_(H)) polypeptides, variable light chain (V_(L)) polypeptides, andCDRs are provided in Table 1 below.

TABLE 1 V_(H), V_(L), and CDR Amino Acid Sequences 1H4/1K4 V_(H)METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVS GFSLSSY (SEQ ID NO: 1)TMGWVRQAPGKGLEWIG DIKTGDR TYYANWAKGRFTISRTSTTVDLKMT SLTTEDTATYFCARVYVSGNDHYDL WGQGTLVTVSS 1H4/1K4 V_(H) CDR1 GFSLSSY (SEQ ID NO: 2)1H4/1K4 V_(H) CDR2 DIKTGDR (SEQ ID NO: 3) 1H4/1K4 V_(H) CDR3ARVYVSGNDHYDL (SEQ ID NO: 4) 1H4/1K4 V_(L)MDTRAPTQLLGLLLLWLPGARCAYDMTQTPASVEVAVGGTVTIKCQAS Q (SEQ ID NO: 5) SISNYLAWYQQKPGQPPKLLIY RAS TLASGVPSRFKGSGRGTEFTLTISGVE CADAATYYC QLGYTYSNVENAFGGGTEVVVK 1H4/1K4 V_(L) CDR1 QSISNY (SEQ ID NO: 6) 1H4/1K4 V_(L) CDR2RAS (SEQ ID NO: 7) 1H4/1K4 V_(L) CDR3 QLGYTYSNVENA (SEQ ID NO: 8)8H1/8K3 V_(H) METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVS GFSLSSY(SEQ ID NO: 1) TMGWVRQAPGKGLEWIG DIKTGDR TYYANWAKGRFTISRTSTTVDLKMTSLTTEDTATYFC ARVYVSGNDHYDL WGQGTLVTVSS 8H1/8K3 V_(H) CDR1 GFSLSSY(SEQ ID NO: 2) 8H1/8K3 V_(H) CDR2 DIKTGDR (SEQ ID NO: 3)8H1/8K3 V_(H) CDR3 ARVYVSGNDHYDL (SEQ ID NO: 4) 8H1/8K3 V_(L)MDTRAPTQLLGLLLLWLPGARCAYDMTQTPASVEVAVGGTVTIKCQAS Q (SEQ ID NO: 9) SISNYLAWYQQKPGQPPKLLIY RAS NLASGVSSRFKGSGRGTEFTLTISGVE CADAATYYC QLGYTYTNVENAFGGGTEVVVK 8H1/8K3 V_(L) CDR1 QSISNY (SEQ ID NO: 6) 8H1/8K3 V_(L) CDR2RAS (SEQ ID NO: 7) 8H1/8K3 V_(L) CDR3 QLGYTYTNVENA (SEQ ID NO: 10)1H2/1K4 V_(H) METGLRWLLLVAVLKGVQCQEQLVESGGGLVQSEGSLTLTCTAS GFSFSS(SEQ ID NO: 11) TK YMCWVRQAPGKRPEWIA CIGTDT TYYASWAKGRFTISRTSSTTVTLQMTSLTAADTATYFC ARSSSTGYYNL WGQGTLVTVSS 1H2/1K4 V_(H) CDR1 GFSFSSTK(SEQ ID NO: 12) 1H2/1K4 V_(H) CDR2 CIGTDT (SEQ ID NO: 13)1H2/1K4 V_(H) CDR3 ARSSSTGYYNL (SEQ ID NO:14) 1H2/1K4 V_(L)MDTRAPTQLLGLLLLWLPGARCADVVMTQTPASVSEPVGGTVTIKCQAS Q (SEQ ID NO: 15)SIRSY LAWYQQKPGQPPKLLIY YAS TLASGVSSRFKGSGSGTEFTLTINGVQ CDDAATYYCHDYYTFTDND FGGGTEVVVK 1H2/1K4 V_(L) CDR1 QSIRSY (SEQ ID NO: 16)1H2/1K4 V_(L) CDR2 YAS (SEQ ID NO: 17) 1H2/1K4 V_(L) CDR3 HDYYTFTDND(SEQ ID NO: 18) 3H1/3K3 V_(H)METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPEGSLTLTCTAS GFSFSS (SEQ ID NO:19) TKYMCWVRQAPGRGPEWVA CIGVGSRGS TYYASRAKGRFTISKTSSTTV TLQMTSLTAADTATYFCARSSTTGYYIL WGQGTLVTVSS 3H1/3K3 V_(H) CDR1 GFSFSSTK (SEQ ID NO: 12)3H1/3K3 V_(H) CDR2 CIGVGSRGS (SEQ ID NO: 20) 3H1/3K3 V_(H) CDR3ARSSTTGYYIL (SEQ ID NO: 21) 3H1/3K3 V_(L)MDTRAPTQLLGLLLLWLPGARCAFEMTQTPSSVSAAVGGTVTIKCQAS ESI (SEQ ID NO: 22) YSYLAWYQQKPGQPPKLLIY KAS TLASGVSSRFKGSGSGTEFTLTISGVQC DDAATYYC QNYYTFTENDVGGGTEVVVK 3H1/3K3 V_(L) CDR1 ESIYSY (SEQ ID NO: 23) 3H1/3K3 V_(L) CDR2KAS (SEQ ID NO: 24) 3H1/3K3 V_(L) CDR3 QNYYTFTEND (SEQ ID NO:25)5H1/5K1 V_(H) METGPRWLLLVAVLKGVQCQEQLAESGGGLVQPEGSLTLTCTAS GFSFW(SEQ ID NO: 26) R YMCWVRQAPGKGLEWVA CIDGGNTNR LYYASWAKGRFTISKTSSTTVTLHMTSLTVADTATYFS ARVRLGNNDYIDL WGQGTLVTVSS 5H1/5K1 V_(H) CDR1 GFSFWR(SEQ ID NO: 27) 5H1/5K1 V_(H) CDR2 CIDGGNTNR (SEQ ID NO: 28)5H1/5K1 V_(H) CDR3 ARVRLGNNDYIDL (SEQ ID NO: 29) 5H1/5K1 V_(L)MDTRAPTQLLGLLLLWLPGARCDVVLTQTPASVEAAVGGTVTIKCQAS QS (SEQ ID NO:30) ISNYLAWYQQKPGQPPKLLIY RAS TLASGVPSRFKGSGSGTQFTLTISDLEC ADAATYYC QQGYNWDLDGAFGGGTEVVVK 5H1/5K1 V_(L) CDR1 QSISNY (SEQ ID NO: 6) 5H1/5K1 V_(L) CDR2RAS (SEQ ID NO: 7) 5H1/5K1 V_(L) CDR3 QQGYNWDLDGA (SEQ ID NO: 31)

In certain embodiments, an antibody of the present disclosure furtherspecifically binds to a metabolite of symmetric dimethylarginine.Metabolites of interest include, but are not limited to, symmetricNα-acetyl-dimethylarginine (Ac-SDMA). According to some embodiments, anantibody of the present disclosure has reactivity of Ac-SDMA of greaterthan 5%, greater than 10%, greater than 15%, greater than 20%, greaterthan 25%, greater than 30%, greater than 35%, greater than 40%, greaterthan 45%, or greater than 50%, of its reactivity for SDMA.

Aspects of the present disclosure further include nucleic acids. Incertain embodiments, a nucleic acid of the present disclosure encodes avariable heavy chain (V_(H)) polypeptide, a variable light chain (V_(L))polypeptide, or both, of any of the antibodies of the presentdisclosure, including but not limited to a V_(H) and/or a V_(L) thatincludes the CDRs of any of the antibodies set forth in Table 1.Examples of nucleic acids having nucleotide sequences that encodeexample antibodies of the present disclosure are provided in Table 2below.

TABLE 2 Nucleotide Sequences 1H4/1K4 V_(H)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTG (SEQ ID NO: 32)TCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATACAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGACATTAAGACTGGTGATAGGACATACTACGCGAACTGGGCAAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCCGAGTGTATGTTAGTGGTAATGATCACTATGACTTGTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGCGGACAGCCGAAA 1H4/1K4 V_(L)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGC (SEQ ID NO: 33)TCCCAGGTGCCAGATGTGCCTATGATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGTATTAGTAACTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGACGTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACTGGGTTATACTTATAGTAATGTTGAGAATGCTTTCGGCGGAGGGACCGAGGTGG TGGTCAAAGGTGATCCCGTG8H1/8K3 V_(H) ATGGAGACCGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTG(SEQ ID NO: 34) TCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATACAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGACATTAAGACTGGTGATAGGACATACTACGCGAACTGGGCAAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCCGAGTGTATGTTAGTGGTAATGATCACTATGACTTGTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGCGGACAGCCGAAA 8H1/8K3 V_(L)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGC (SEQ ID NO: 35)TCCCAGGTGCCAGATGTGCCTATGATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAACTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCAATCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGACGTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACTGGGTTATACTTATACTAATGTTGAGAATGCTTTCGGCGGAGGGACCGAGGTGGT GGTCAAAGGTGATCCCGTG1H2/1K4 V_(H) ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTG(SEQ ID NO: 36) TCCAGTGTCAGGAGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCCAGTCTGAGGGATCCCTGACACTCACCTGCACAGCTTCTGGATTCTCCTTCAGCAGCACCAAGTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGAGGCCTGAGTGGATCGCATGCATTGGTACTGATACCACTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGAAGTAGTAGTACTGGTTATTATAATTTGTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGCGGACAGCCGAAA 1H2/1K4 V_(L)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGC (SEQ ID NO: 37)TCCCAGGTGCCAGATGTGCCGACGTCGTGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTCGTAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAACGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCACGACTATTATACTTTTACTGATAATGATTTCGGCGGAGGGACCGAGGTGGTGGTC AAAGGTGATCCCGTG3H1/3K3 V_(H) ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTG(SEQ ID NO: 38) TCCAGTGTCAGGAGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGACACTCACCTGCACAGCTTCTGGATTCTCCTTCAGCAGCACCAAGTACATGTGCTGGGTCCGCCAGGCTCCAGGGAGGGGGCCTGAGTGGGTCGCATGTATTGGTGTTGGTAGTCGTGGTAGCACTTACTACGCGAGCCGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGGAGTAGTACTACTGGTTATTATATTTTATGGGGCCAGGGCACCCTGGTCACCGTCTCGAGCGGACAGCCGAAA 3H1/3K3 V_(L)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGC (SEQ ID NO: 39)TCCCAGGTGCCAGGTGTGCATTCGAGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGAGCATTTACAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGAAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAAACTATTATACTTTTACTGAGAATGATGTCGGCGGAGGGACCGAGGTGGTGGTCA AAGGTGATCCCGTG5H1/5K1 V_(H) ATGGAGACTGGGCCGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTG(SEQ ID NO: 40) TCCAGTGTCAGGAGCAGCTGGCGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCTTCTGGCGCTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCATGTATTGATGGTGGCAATACTAATAGGCTCTATTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCACATGACCAGTCTGACAGTCGCGGACACGGCCACCTATTTCAGTGCGAGAGTTCGGCTTGGTAATAATGATTATATAGACTTGTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGCGGACAGCCGAAA 5H1/5K1 V_(L)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGC (SEQ ID N0: 41)TCCCAGGTGCCAGATGTGATGTTGTGCTGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTAACTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGGTTATAATTGGGATCTTGATGGTGCTTTCGGCGGAGGGACCGAGGTGGTGGTC AAAGGTGATCCCGTG

Also provided are expression vectors that include any of the nucleicacids of the present disclosure. The expression vectors find use, e.g.,for expressing a V_(H) and/or a V_(L) of an antibody of the presentdisclosure in a host cell. The expression of natural or syntheticnucleic acids encoding a V_(H) and/or a V_(L) of an antibody of thepresent disclosure will typically be achieved by operably linking anucleic acid encoding the V_(H) and/or V_(L) to a promoter (which iseither constitutive or inducible), and incorporating the construct intoan expression vector. The vectors can be suitable for replication andintegration in prokaryotes, eukaryotes, or both. Typical cloning vectorscontain transcription and translation terminators, initiation sequences,and promoters useful for regulation of the expression of the nucleicacid encoding the V_(H) and/or V_(L). The vectors optionally comprisegeneric expression cassettes containing at least one independentterminator sequence, sequences permitting replication of the cassette inboth eukaryotes and prokaryotes, i.e., shuttle vectors, and selectionmarkers for both prokaryotic and eukaryotic systems. See Sambrook et al(1989). To obtain high levels of expression of a cloned nucleic acid itis common to construct expression plasmids which typically contain astrong promoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.

Accordingly, aspects of the present disclosure further include cells,e.g., recombinant host cells. In certain embodiments, provided are cellsthat include any of the nucleic acids and/or expression vectors of thepresent disclosure. According to some embodiments, provided are cellsthat include a first nucleic acid encoding a variable heavy chain(V_(H)) polypeptide of an antibody of the present disclosure, and asecond nucleic acid encoding a variable light chain (V_(L)) polypeptideof the antibody. In certain embodiments, provided are cells that includea first expression vector comprising the first nucleic acid, and asecond expression vector comprising the second nucleic acid. Cells ofthe present disclosure may be produced by introducing one or morenucleic acids and/or expression vectors of the present disclosure intohost cells via methods known in the art, e.g., electroporation,lipofection, microinjection, or the like.

Also provided are methods of making the antibodies of the presentdisclosure. In certain embodiments, such methods include culturing acell (e.g., recombinant host cell) of the present disclosure underconditions suitable for the cell to express the antibody, wherein theantibody is produced. The suitable conditions for culturing the cellsuch that the antibody is expressed may vary. Such conditions mayinclude culturing the cell in a suitable container (e.g., a cell cultureplate or well thereof), in suitable medium (e.g., cell culture medium,such as DMEM, RPMI, MEM, IMDM, DMEM/F-12, or the like) at a suitabletemperature (e.g., 32° C.-42° C., such as 37° C.) and pH (e.g., pH7.0-7.7, such as pH 7.4) in an environment having a suitable percentageof CO₂, e.g., 3% to 10%, such as 5%).

Also provided are methods of preparing polyclonal antibodies thatspecifically bind any of the new immunogens set forth in Formula 1.Antiserum containing antibodies is obtained by well-establishedtechniques involving immunization of an animal, such as rabbits andsheep, with an appropriate immunogen set forth in Formula 1 andobtaining antisera from the blood of the immunized animal after anappropriate waiting period. Reviews are provided by Parker,Radioimmunoassay of Biologically Active Compounds, Prentice-Hall(Englewood Cliffs, N.J., U.S., 1976), Butler, J. Immunol. Meth. 7: 124(1975); Broughton and Strong, Clin. Chem. 22: 726 732 (1976); andPlayfair, et al., Br. Med. Bull. 30: 24 31 (1974). The immunizationprocedures are well established in the art and are set forth in numeroustreatises and publications including “The Immunoassay Handbook”, 2ndEdition, edited by David Wild (Nature Publishing Group, 2000) and thereferences cited therein. The degree of the antibody purificationrequired depends on the desired application. For many purposes there isno requirement for purification.

Serum harvested may be tested for the presence of antibodies thatspecifically bind symmetrically dimethylated arginine analyte using asymmetric dimethylarginine protein conjugate or other symmetricdimethylarginine conjugates in either an ELISA format or homogeneousenzyme immunoassay format. This technique is generally applicable toproduce polyclonal antibodies to symmetrically dimethylated arginineanalyte as described herein and to assess their utility. The specificantibodies prepared are useful as reagents for immunoassays for thedetection or determination (optionally including quantification) ofsymmetric dimethylarginine.

The following procedure may be employed to prepare monoclonalantibodies, in particular for monoclonal antibodies that specificallybind the immunogens of Formula 1. Monoclonal antibodies may be producedaccording to the standard techniques of Kohler and Milstein, Nature265:495 497, 1975. Reviews of monoclonal antibody techniques are foundin Lymphocyte Hybridomas, ed. Melchers, et al. Springer-Verlag (New York1978), Nature 266: 495 (1977), Science 208: 692 (1980), and Methods ofEnzymology 73 (Part B): 3 46 (1981). Samples of an appropriate immunogenpreparation are injected into an animal such as a rabbit or mouse and,after a sufficient time, the animal is sacrificed and spleen cellsobtained. Alternatively, the spleen cells of a non-immunized animal canbe sensitized to the immunogen in vitro. The spleen cell chromosomesencoding the base sequences for the desired immunoglobulins can becompressed by fusing the spleen cells, generally in the presence of anon-ionic detergent, for example, polyethylene glycol, with a myelomacell line. The resulting cells, which include fused hybridomas, areallowed to grow in a selective medium, such as HAT-medium, and thesurviving immortalized cells are grown in such medium using limitingdilution conditions. The cells are grown in a suitable container, e.g.,microtiter wells, and the supernatant is screened for monoclonalantibodies having the desired specificity. Various techniques exist forenhancing yields of monoclonal antibodies, such as injection of thehybridoma cells into the peritoneal cavity of a mammalian host, whichaccepts the cells, and harvesting the ascites fluid. Where aninsufficient amount of the monoclonal antibody collects in the ascitesfluid, the antibody may be harvested from the blood of the host.Alternatively, the cell producing the desired antibody can be grown in ahollow fiber cell culture device or a spinner flask device, both ofwhich are well known in the art. Various conventional ways exist forisolation and purification of the monoclonal antibodies from otherproteins and other contaminants (see Kohler and Milstein, supra).

The following procedure may be employed to prepare recombinantmonoclonal antibodies, in particular monoclonal antibodies thatspecifically bind the immunogens of Formula 1. Single B-cell screen,cloning and expression was performed. Peripheral blood mononuclear cells(PBMCs) were isolated from whole blood of rabbit and cultured the sameday and plating single B-cells onto 40×96 well plates. The 40×96-wellplates were incubated at 37° C./5% CO₂ for seven days in B cellculturing media and the supernatants were then screened by indirectELISA against SDMA-SBAP-BSA antigen to determine antigen-positive wells.Antigen-positive wells were preserved in RNA lysis buffer and stored at−80° C. mRNA was isolated from selected B cell well (SDMA-SBAP-BSAantigen-positive wells) by Dynabeads mRNA DIRECT purification kit(Ambion, catalog #61012). cDNA was synthesized and 2 rounds of PCRperformed to prepare the antibody variable region cDNA for cloning.Rabbit IgG heavy and kappa light chain variable region cDNAs were clonedinto mammalian expression vectors with a rabbit heavy and a light chainconstant region, respectively. Expression constructs were co-transfectedinto HEK 293 cells and cell culture supernatants assayed by indirectELISA against SDMA-SBAP-BSA antigen. The antibodies were purifiedaccording to standard approaches for antibody purification fromsupernatants. In general, antibodies can be purified by known techniquessuch as chromatography, e.g., DEAE chromatography, ABx chromatography,and the like, filtration, and so forth. Antibodies may be screened usingany of several techniques, for example using a homogeneous enzymeimmunoassay format as illustrated in FIG. 10, and considering suchproperties as, conjugate inhibition, curve size and cross-reactivity,and so forth.

DNA sequencing was performed for selected positive rabbit monoclonalantibodies. The rabbit IgG heavy chain sequence is approximately 1200 bpand can be sequenced from the 5′ ends to obtain a reliable full-lengthvariable sequence. The rabbit kappa light chain is approximately 700 bpand full-length variable sequence can be reliably obtained fromsequencing in the 5′ direction. All heavy chain and kappa chain variableregion sequences were translated. The resulting amino acid sequences ofthe V_(H) and V_(L) of example antibodies are provided in Table 1 above.

Compositions

The present disclosure also provides compositions. According to someembodiments, a composition of the present disclosure includes any of thecompounds, antibodies, nucleic acids, expression vectors, and/or cellsof the present disclosure.

In certain aspects, a composition of the present disclosure includes anyof the compounds, antibodies, nucleic acids, expression vectors, and/orcells of the present disclosure present in a liquid medium. The liquidmedium may be an aqueous liquid medium, such as water, a bufferedsolution, or the like. One or more additives such as a salt (e.g., NaCl,MgCl₂, KCl, MgSO₄), a buffering agent (a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), asolubilizing agent, a detergent (e.g., a non-ionic detergent such asTween-20, etc.), a nuclease inhibitor, a protease inhibitor, glycerol, achelating agent, and the like may be present in such compositions.

In certain embodiments, the compositions of the present disclosure finduse as reagents in performing any of the immunoassays of the presentdisclosure, including any of the homogenous enzyme immunoassaysdescribed herein. For example, a composition that includes any of thecompounds of Formula 1 of the present disclosure, any of the antibodiesof the present disclosure, or both, may be employed to perform suchimmunoassays. According to some embodiments, the reagents are providedin lyophilized form, e.g., to increase stability, convenience, and/orthe like. As just one example, a composition that includes any of thecompounds of Formula 1 of the present disclosure, any of the antibodiesof the present disclosure, or both, may be provided in the form oflyophilized reagent spheres as described in U.S. Pat. No. 5,413,732, thedisclosure of which is incorporated herein by reference in its entiretyfor all purposes. Briefly, such spheres may be made, e.g., by forming ahomogeneous solution of a reagent; measuring uniform drops of thesolution (e.g., 2 to 50 μL); dispensing the uniform, measured drops intoan unagitated cryogenic liquid (e.g., liquid nitrogen), whereby thedrops are frozen; collecting the frozen drops from the cryogenic liquid;and lyophilizing the frozen drops, thereby forming a plurality oflyophilized reagent spheres.

Immunoassays

Aspects of the present disclosure further include methods of using anyof the compounds of Formula 1 of the present disclosure and/or any ofthe antibodies of the present disclosure. In certain embodiments, suchcompounds and/or antibodies may be used for detecting (includingdetermining an amount of) at least one symmetrically dimethylatedarginine analyte in a medium, e.g., a medium that includes a biologicalsample of interest. For example, according to some embodiments, providedare methods for determining an amount of at least one symmetricallydimethylated arginine analyte in a medium, the methods includingcombining in a medium a sample suspected of containing at least onesymmetrically dimethylated arginine analyte, and any of the antibodiesof the present disclosure. Such methods further include determining thepresence or absence of a complex comprising the symmetricallydimethylated arginine analyte and the antibody, wherein the presence ofthe complex indicates the presence of the symmetrically dimethylatedarginine analyte in the sample. In certain embodiments, the mediumfurther includes any of the compounds of Formula 1 of the presentdisclosure. For example, the medium may further include a symmetricdimethylarginine conjugate that has a symmetric dimethylarginine moietyand a detectable label (e.g., enzyme, such as G6PDH).

The sample suspected of containing at least one symmetricallydimethylated arginine analyte may be any sample of interest. In certainembodiments, the sample is whole blood, blood serum, blood plasma,urine, sputum, semen, saliva, ocular lens fluid, cerebral fluid, spinalfluid, amniotic fluid, tissue culture media, or the like, and includingdilutions thereof.

The present disclosure provides immunoassay methods for assessing thepresence or absence of a symmetrically dimethylated arginine analyte ina sample suspected of containing the analyte. Immunoassays of thepresent disclosure can be of a variety of formats. The immunoassays maybe separation immunoassays (also known as heterogeneous immunoassays) orhomogeneous immunoassays. Furthermore, the immunoassays may bequalitative or quantitative. Assays of this disclosure include bothsandwich and competition assays. The immunoassays may embody other typesof assays that are neither sandwich nor competition assays, as incertain assays involving immunoprecipitation. In certain embodiments,the immunoassay is a homogeneous immunoassay, where the assay reagentsand sample are mixed together to form a homogeneous assay mixture.

In certain embodiments, the immunoassay is a homogeneous enzymeimmunoassay system used for the analysis of a symmetrically dimethylatedarginine analyte in a biological fluid sample. In some instances, theimmunoassay is based on competition between the symmetricallydimethylated arginine analyte in the sample and labeled Nα-acylated-SDMAand/or Nα-alkylated-SDMA for antibody binding sites. In someembodiments, the label is a protein, such as an enzyme. For example, thelabel may be an enzyme the activity of which may be measuredspectrophotometrically. In one non-limiting example, an assay of thepresent disclosure employs Nα-acylated-SDMA and/or Nα-alkylated-SDMAlabeled with the enzyme glucose-6-phosphate dehydrogenase (G6PDH) forantibody binding sites. In certain embodiments, enzyme activitydecreases upon binding to the antibody, such that the concentration ofthe symmetrically dimethylated arginine analyte in the sample can bemeasured in terms of enzyme activity. In some cases, active enzymeconverts nicotinamide adenine dinucleotide (NAD⁺) to NADH, resulting inan absorbance change that is measured spectrophotometrically. In certaininstances, endogenous serum G6PDH does not interfere with theimmunoassay because the coenzyme NAD⁺ functions only with the bacterial(Leuconostoc mesenteroides) enzyme employed in the assay.

In general, the immunoassays of the present disclosure for detecting thepresence (or absence) of a symmetrically dimethylated arginine analytein a sample can be conducted by adding, to a reaction mixture, (i) asample suspected of containing a symmetrically dimethylated arginineanalyte and (ii) an antibody that specifically binds to a symmetricallydimethylated arginine analyte to form a complex between the antibody andsymmetrically dimethylated arginine analyte that may be present in thesample. The method also includes detecting the presence or absence ofthe complex. The presence (or absence) of the complex may be indicativeof the presence (or absence) of symmetrically dimethylated arginineanalyte in the sample. Moreover, the amount of complex formed can beassessed to determine the concentration of symmetrically dimethylatedarginine analyte present in the sample (e.g., to provide an assessmentof serum or tissue concentration of symmetrically dimethylated arginineanalyte in a subject from whom the sample was obtained). The presenceand/or amount of complex can be assessed directly (e.g., by detectingbound antibody in the complex) or indirectly (e.g., by assessingactivity of an enzyme in a symmetric dimethylarginine enzyme conjugate,where when the symmetric dimethylarginine enzyme conjugate is not boundto antibody, a detectable signal is generated, indicating that thesymmetrically dimethylated arginine analyte antibody in the reactionmixture has been bound by symmetrically dimethylated arginine analytefrom the sample) (see, e.g., FIG. 10).

In general, the immunoassays of the present disclosure entail combiningin a medium (e.g., assay medium or assay reaction mixture), the samplewith a symmetrically dimethylated arginine analyte antibody underconditions that permit the formation of a stable complex between theanalyte in the sample and the antibody.

Assays may be performed in solution or may use a solid (insoluble)support (e.g., polystyrene, nitrocellulose, or beads), using anystandard methods (e.g., as described in Current Protocols in Immunology,Coligan et al., ed.; John Wiley & Sons, New York, 1992). Such methodsinclude ELISAs (enzyme-linked immunosorbent assays), IRMAs(immunoradiometric assays), and RIAs (radioimmunoassays). In certainembodiments, the assay is performed in solution, e.g., the assay isperformed without the assay reagents attached to or associated with asolid support.

Where the assay is performed in solution, the test sample (and,optionally a control sample) may be incubated with an anti-symmetricallydimethylated arginine analyte antibody for a time period sufficient toallow formation of analyte and affinity reagent complexes. As previouslynoted, the symmetrically dimethylated arginine analyte antibody mayinclude a detectable label, e.g., radionuclide, fluorescer, or enzyme.The sample may then be treated to separate the symmetricallydimethylated arginine analyte antibody complexes from excess, unreactedsymmetrically dimethylated arginine analyte antibody (e.g., by additionof a secondary antibody (e.g., anti-immunoglobulin antiserum)) followedby centrifugation to precipitate the secondary complexes, or by bindingto an affinity surface such as a second, unlabeled antibody fixed to asolid substrate such as Sepharose® or a plastic well). Detection ofsymmetrically dimethylated arginine analyte antibody bound to asymmetrically dimethylated arginine analyte may be achieved in a varietyof ways. If necessary, a substrate for the detectable label may be addedto the sample.

Where the assay uses a solid support, the support can have asymmetrically dimethylated arginine analyte antibody (or conjugate)bound to a support surface. Binding of the assay reagent may facilitatethe stable, wash-resistant binding of symmetrically dimethylatedarginine analyte which may be present in the sample (or antibody that isnot bound to symmetrically dimethylated arginine analyte from thesample, and is present in the reaction mixture, as in a competitivebinding assay) to the solid support via specific binding to theantibody. The insoluble support may be any composition to whichantibodies or suitable symmetric dimethylarginine conjugates can bebound, which can be separated from soluble material, and which isotherwise compatible with the overall method of detection ofsymmetrically dimethylated arginine analyte in a sample.

The surface of such supports may be solid or porous and of anyconvenient shape. Examples of suitable insoluble supports to which thesymmetrically dimethylated arginine analyte antibody or symmetricallydimethylarginine conjugate is bound include beads, e.g., magnetic beads,membranes and microtiter plates. These can be composed of glass, plastic(e.g., polystyrene), polysaccharides, nylon or nitrocellulose.

Assay reagents can include the symmetrically dimethylated arginineanalyte antibody as disclosed herein, as well as secondary antibodies,which may be optionally detectably labeled. After binding of an assayreagent to the support, the support may be treated with a blockingagent, which binds to the support in areas not occupied by the assayreagent. Suitable blocking agents include non-interfering proteins suchas bovine serum albumin, casein, gelatin, and the like. Alternatively,detergents at non-interfering concentrations, such as Tween, NP40,TX100, and the like may be used. Such blocking treatment may reducenonspecific binding.

Assays of the present disclosure include both qualitative andquantitative assays. Typical quantitative methods involve mixing ananalyte with a pre-determined amount of the reagent antibody, andcorrelating the amount of complex formed with the amount of analyte inthe original sample using a relationship determined using standardsamples containing known amounts of analyte in the range expected forthe sample to be tested. In a qualitative assay, sufficient complexabove or below a threshold level established by samples known to containor be free of analyte can be used to establish the assay result. Unlessotherwise stated, the act of “measuring” or “determining” in thisdisclosure encompasses both qualitative and quantitative determination.

Immunoassay reagents that find use alone or in combination in the assaysdescribed herein include, but are not limited to, a symmetricallydimethylated arginine analyte antibody, a symmetric dimethylarginineconjugate, and a symmetrically dimethylated arginine analyte (e.g., as acontrol or in competitive binding assays). Immunoassay reagents can beprovided in a buffered aqueous solution. Such solutions may includeadditional components such as surface active additives, organicsolvents, defoamers, buffers, surfactants, and anti-microbial agents.Surface active additives can be introduced to maintain hydrophobic orlow-solubility compounds in solution, and stabilize components in thesolution. Examples include bulking agents such as β-lactoglobulin (BLG)or polyethyleneglycol (PEG); defoamers and surfactants such as Tween-20,Plurafac A38, Triton X-100, Pluronic 25R2, rabbit serum albumin (RSA),bovine serum albumin (BSA), and carbohydrates. Examples of organicsolvents can include methanol and other alcohols. Various buffers may beused to maintain the pH of the solution during storage. Illustrativebuffers include HEPES, borate, phosphate, carbonate, tris, barbital andthe like. Anti-microbial agents also extend the storage life of theimmunoassay reagent.

The symmetric dimethylarginine conjugates and/or the symmetricallydimethylated arginine analyte antibodies to be used as reagents in anassay can be insolubilized by attachment to a solid support. This canbe, for example, a wall of a vessel containing the reagent, to aparticulate, or to a large molecular weight carrier that can be kept insuspension but removable by physicochemical means, such ascentrifugation or microfiltration. In some cases, the attachment isthrough one or more covalent bonds. The attachment need not be covalent,but is at least of sufficient strength and/or permanence to withstand aseparation technique (including a wash) that may be part of the assayprocedure. In some cases, the solid support may be functionalized toinclude a reactive group to facilitate attachment of the symmetricdimethylarginine conjugate and/or the symmetrically dimethylatedarginine analyte antibody to the solid support. Nonlimiting examples ofreactive groups that may be used include —COOH, —NH₂, —C(O)H, —SH, andthe like. In some embodiments, the symmetric dimethylarginine conjugateand/or the symmetrically dimethylated arginine analyte antibody can beconjugated to a protein carrier and the protein carrier can beconjugated to the solid support, thus indirectly attaching the symmetricdimethylarginine conjugate and/or the symmetrically dimethylatedarginine analyte antibody to the solid support. Certain particulatematerials include, but are not limited to, agarose, polystyrene,cellulose, polyacrylamide, latex particles, magnetic particles, andfixed red cells. Examples of commercially available matrices include,but are not limited to, Sepharose® (Pharmacia), Poros® resins (RocheMolecular Biochemicals), Actigel Superflow™ resins (SterogeneBioseparations Inc.), and Dynabeads™ (Dynal Inc.). In certainembodiments, the choice of the solid support may depend on one or moreof stability, capacity, accessibility of the coupled antibody, flow rate(or the ability to disperse the resin in the reaction mixture), and easeof separation.

As noted above, immunoassays for detection of a symmetricallydimethylated arginine analyte can be of a variety of formats. Ingeneral, the immunoassays involve combining one or more immunoassayreagents (e.g., at least an anti-symmetrically dimethylated arginineanalyte antibody) with a test sample (i.e., a sample suspected ofcontaining a symmetrically dimethylated arginine analyte) in a medium(e.g., a reaction mixture or an assay mixture). “Reaction mixture” or“assay mixture” generally refers to the combination of a samplesuspected of containing a symmetrically dimethylated arginine analyteand one or more immunoassay reagents as exemplified in the presentdisclosure to facilitate detection of the presence or absence of asymmetrically dimethylated arginine analyte in the sample, where thedetection may be qualitative or quantitative. The reaction mixture isusually an aqueous solution, although the immunoassay reagent(s) may bein solution or immobilized on a support (e.g., a substrate, such as abead). The reaction mixture can include other components compatible withthe immunoassay, e.g., buffers, reagents, and the like.

Immunoassays usually are classified in one of several ways. For example,immunoassays can be classified according to the mode of detection used,i.e., enzyme immunoassays, radio immunoassays, fluorescence polarizationimmunoassays, chemiluminescence immunoassays, turbidimetric assays, etc.Another grouping method is according to the assay procedure used, i.e.,competitive assay formats, sandwich-type assay formats as well as assaysbased on precipitation or agglutination principles. In certaininstances, a further distinction is made depending on whether washingsteps are included in the procedure (so-called heterogeneous assays) orwhether reaction and detection are performed without a washing step(so-called homogeneous assays). Certain assays are described in moredetail below.

Immunoassays may be described as heterogeneous or homogeneous.“Homogeneous immunoassay”, as used herein, refers to an assay methodwhere the complex is not separated from unreacted reaction components,but instead the presence of the complex is detected by a property whichat least one of the reactants acquires or loses as a result of beingincorporated into the complex. Homogeneous assays include systemsinvolving fluorochrome and fluorochrome quenching pairs on differentreagents; enzyme and enzyme inhibitor pairs on different reagents;chromophore and chromophore modifier pairs on different reagents; andlatex agglutination assays.

A certain homogeneous assay is the quantitative homogeneous enzymeimmunoassay in which a symmetric methylarginine is conjugated to anactive enzyme. In some embodiments, the conjugation is arranged so thatthe binding of a symmetrically dimethylated arginine analyte antibody tothe symmetric methylarginine conjugate affects enzymatic activity of theconjugate in a qualitative or quantitative fashion. If a samplecontaining symmetrically dimethylated arginine analyte is premixed withthe antibody, the antibody may complex with the symmetricallydimethylated arginine analyte and thus be prevented from binding to theenzyme conjugate. In this way, the activity of the enzyme in theconjugate can be correlated with the amount of symmetricallydimethylated arginine analyte present in the sample.

G6PDH is a certain enzyme useful in such assays. In some embodiments,the G6PDH is a variant of a naturally-occurring G6PDH in which one ormore lysine residues are deleted or substituted, or one or more cysteineresidues are introduced. For example, Leuconostoc mesenteroides G6PDHare dimeric enzymes that have the ability to catalyze the oxidation ofD-glucose-6-phosphate to D-glucono-delta-lactone-6-phosphate byutilizing either NAD⁺ or NADP⁺. This property of using NAD⁺differentiates these enzymes from human G6PDH, which utilizes only NADP⁺effectively, and allows L. mesenteroides-specific G6PDH activity to bemeasured in the presence of human G6PDH, as for example in human-derivedsamples. Two certain genera of bacteria from which to select G6PDH areLeuconostoc and Zymomonas. Within these genera L. mesenteroides, L.citreum, L. lactis, L. dextranicum, and Z. mobilis are of interest,where L. mesenteroides, L. citreum, and L. lactis are specific examples.Another example of a homogeneous assay system is the cloned enzyme donorimmunoassay.

Symmetric dimethylarginine derivatives with thiol reactive groups can beprepared as described above, and may be allowed to react with aglucose-6-phosphate dehydrogenase (G6PDH) mutant enzyme to form therespective enzyme conjugates (see, e.g., FIG. 7). The mutant enzyme maybe obtained by the procedure described in U.S. Pat. Nos. 6,090,567 and6,033,890, the disclosures of which are incorporated herein byreference.

In some embodiments, the immunoassay further includes adding a symmetricdimethylarginine conjugate that has a symmetric dimethylarginine moietyand a detectable label to the sample. The presence or absence ofsymmetrically dimethylated arginine analyte in the sample can bedetected by detecting the detectable label. The detectable label mayinclude an enzyme and the detecting may be performed by assayingactivity of the enzyme. In certain embodiments, the enzyme is adehydrogenase, such as G6PDH.

Luminescence oxygen channeling assay (LOCI) is a chemilumenscencehomogeneous immunoassay whereby a biotinylated antibody to the analytebinds to streptavidin-coated donor beads and a second antibody to theanalyte is directly conjugated to LOCI acceptor beads. In the presenceof the analyte, the two beads come into close proximity. The excitationof the donor beads at 680 nm generates singlet oxygen molecules thattrigger a series of chemical reactions in the LOCI acceptor beadsresulting in a detectable peak of light emission at 615 nm (Ullman, E.F. et al. (1996) Clin. Chem. 42, 1518-1526).

In a separation-based or “heterogeneous” assay, the detecting of acomplex of a symmetrically dimethylated arginine analyte antibody and ananalyte involves a process where the complex formed is physicallyseparated from either unreacted analyte, unreacted antibody, or both.

In a heterogeneous immunoassay, a complex of an antibody and asymmetrically dimethylated arginine analyte may be first formed in thefluid phase, and then subsequently captured by a solid phase reagent orseparated on the basis of an altered physical or chemical property, suchas by gel filtration or precipitation. Alternatively, one of thereagents may be attached to a solid phase before contacting with otherreagents, and then the complex may be recovered by washing the solidphase free of unreacted reagents. Separation-based assays typicallyinvolve use of a labeled derivative or labeled antibody to facilitatedetection or quantitation of the complex. Suitable labels includeradioisotopes such as ¹²⁵I, enzymes such as peroxidase andβ-galactosidase, and fluorescent labels such as fluoresceinisothiocyanate. The separation step involves removing labeled reagentpresent in complex form from unreacted labeled reagent. The amount oflabel in the complex can be measured directly or inferred from theamount left unreacted.

Assays of the present disclosure include both sandwich and competitionassays. Sandwich assays typically involve forming a complex in which theanalyte to be measured is sandwiched between one reagent, such as afirst antibody used ultimately for separation of the complex, andanother reagent, such as a second antibody used as a marker for theseparated complex. Competition assays involve a system in which theanalyte to be measured competes with a derivative of the analyte forbinding to another reagent, such as an antibody. An example of acompetition assay using EMIT® is described in U.S. Pat. No. 3,817,837.

The compounds and methods of the presently disclosed embodiments alsoencompass the use of these materials in lateral flow chromatographytechnologies. Lateral flow chromatography involves a membrane stripwhich includes a detection device, such as a non-isotopic signalgenerating moiety, for symmetrically dimethylated arginine analyte. Asample from a patient may then be applied to the membrane strip. Thesample may interact with the detection device, producing a result. Theresults can signify several things, including the absence of thesymmetrically dimethylated arginine analyte in the sample, the presenceof the symmetrically dimethylated arginine analyte in the sample, and/orthe concentration of the symmetrically dimethylated arginine analyte inthe sample.

Certain embodiments provide a method of qualitatively determining thepresence or absence of a symmetrically dimethylated arginine analyte ina sample, through the use of lateral flow chromatography. In certainembodiments, the basic design of the qualitative lateral flow device isas follows: 1) The sample pad is where the sample is applied. The samplepad is treated with chemicals such as buffers or salts, which, whenre-dissolved, optimize the chemistry of the sample for reaction with theconjugate, test, and control reagents; 2) Conjugate release pad istypically a polyester or glass fiber material that is treated with aconjugate reagent such as an antibody colloidal gold conjugate. Atypical process for treating a conjugate pad is to use impregnationfollowed by drying. In use, the liquid sample added to the test willre-dissolve the conjugate so that it will flow into the membrane; 3) Themembrane substrate is usually made of nitrocellulose or a similarmaterial whereby antibody capture components are immobilized; 4) Awicking pad is used in tests where blood plasma must be separated fromwhole blood. An impregnation process is usually used to treat this padwith reagents intended to condition the sample and promote cellseparation; 5) The absorbent pad acts as a reservoir for collectingfluids that have flowed through the device; and 6) The above layers andmembrane system are laminated onto a plastic backing with adhesivematerial which serves as a structural member.

Certain embodiments provide a method of qualitatively determining thepresence of a symmetrically dimethylated arginine analyte in a sample,through the use of lateral flow chromatography. In these embodiments,the membrane strip includes a sample pad, which is a conjugate releasepad that has an antibody that is specific for the symmetricallydimethylated arginine analyte. This antibody may be conjugated to anon-isotopic signal-generating moiety, such as a colloidal goldparticle. Other detection moieties useful in a lateral flowchromatography environment include dyes, colored latex particles,fluorescently labeled latex particles, non-isotopic signal generatingmoieties, etc. In some instances, the membrane strip further includes acapture line, in which the symmetrically dimethylated arginine analyteantigen or symmetrically dimethylarginine conjugate is immobilized onthe strip. In some embodiments, this immobilization is through covalentattachment to the membrane strip, optionally through a linking group. Inother embodiments, the immobilization is through non-covalent attachmentto the membrane strip. In still other embodiments, the immobilesymmetrically dimethylated arginine analyte in the capture line isattached to a reactive partner, such as an immunogenic carrier like BSA.

Sample from a patient may be applied to the sample pad, where it cancombine with the antibody in the conjugate release pad, thus forming asolution. This solution may then migrate chromatographically bycapillary action across the membrane. When symmetrically dimethylatedarginine analyte is present in the sample, a symmetrically dimethylatedarginine analyte antibody complex may be formed, which migrates acrossthe membrane by capillary action. When the solution reaches the captureline, the symmetrically dimethylated arginine analyte antibody complexmay compete with the immobile symmetrically dimethylated arginineanalyte for the limited binding sites of the antibody. When a sufficientconcentration of symmetrically dimethylated arginine analyte is presentin the sample, it may fill the limited antibody binding sites. Incertain instances, this will prevent the formation of a coloredantibody-immobile symmetrically dimethylated arginine analyte complex inthe capture line. Therefore, absence of color in the capture lineindicates the presence of symmetrically dimethylated arginine analyte inthe sample.

In the absence of symmetrically dimethylated arginine analyte in thesample, a colored antibody-immobile symmetrically dimethylated arginineanalyte complex may form once the solution reaches the capture line ofthe membrane strip. In some instances, the formation of this complex inthe capture line is evidence of the absence of symmetricallydimethylated arginine analyte in the sample.

Certain embodiments provide a method of quantitatively determining theamount of a symmetrically dimethylated arginine analyte in a sample,through the use of lateral flow chromatography. This technology isfurther described in U.S. Pat. Nos. 4,391,904; 4,435,504; 4,959,324;5,264,180; 5,340,539; and 5,416,000, the disclosures of which areincorporated herein by reference. In some embodiments, the antibody maybe immobilized along the entire length of the membrane strip. Ingeneral, if the membrane strip is made from paper, the antibody may becovalently bound to the membrane strip. If the membrane strip is madefrom nitrocellulose, then the antibody can be non-covalently attached tothe membrane strip through, for example, hydrophobic and electrostaticinteractions. The membrane strip may include a conjugate release padthat includes the symmetrically dimethylated arginine analyte attachedto a detector moiety. In certain embodiments, the detector moiety is anenzyme, such as horseradish peroxidase (HRP).

In certain embodiments, sample from a patient is applied to the membranestrip, where it can combine with the symmetrically dimethylated arginineanalyte/detector molecule in the conjugate release pad, thus forming asolution. This solution may then be allowed to migratechromatographically by capillary action across the membrane. Whensymmetrically dimethylated arginine analyte is present in the sample,both the sample symmetrically dimethylated arginine analyte and thesymmetrically dimethylated arginine analyte/detector molecule competefor the limited number of binding sites of the antibody. When asufficient concentration of symmetrically dimethylated arginine analyteis present in the sample, it may fill the limited antibody bindingsites. In some instances, this forces the symmetrically dimethylatedarginine analyte/detector molecule to continue to migrate in themembrane strip. The shorter the distance of migration of thesymmetrically dimethylated arginine analyte/detector molecule in themembrane strip, the lower the concentration of symmetricallydimethylated arginine analyte in the sample, and vice versa. When thesymmetrically dimethylated arginine analyte/detector molecule includesan enzyme, the length of migration of the symmetrically dimethylatedarginine analyte/detector molecule can be detected by applying an enzymesubstrate to the membrane strip. Detection of the product of the enzymereaction may then be utilized to determine the concentration of thesymmetrically dimethylated arginine analyte in the sample. In certainembodiments, the enzyme's color producing substrate such as a modifiedN,N-dimethylaniline is immobilized to the membrane strip and3-methyl-2-benzothiazolinone hydrazone is passively applied to themembrane, thus alleviating the need for a separate reagent to visualizethe color producing reaction.

Fluorescence polarization immunoassay (FPIA) technology is based uponcompetitive binding. FPIA technology is described in, for example, U.S.Pat. Nos. 4,593,089, 4,492,762, 4,668,640, and 4,751,190, thedisclosures of which are incorporated herein by reference.

The FPIA technology can be used to identify the presence ofsymmetrically dimethylated arginine analyte and can be used in assaysthat quantify the amount of symmetrically dimethylated arginine analytein a sample. In part, the rotational properties of molecules in solutionallow for the degree of polarization to be directly proportional to thesize of the molecule. Accordingly, polarization may increase asmolecular size increases. That is, when linearly polarized light is usedto excite a fluorescent-labeled or other luminescent-labeledsymmetrically dimethylated arginine analyte thereof, which is small androtates rapidly in solution, the emitted light may be significantlydepolarized. When the fluorescent-labeled symmetrically dimethylatedarginine analyte interacts with or is bound to an antibody, the rotationmay be slowed and the emitted light may be highly polarized. In somecases, this is because the antibody significantly and measurablyincreases the size of the complex. Also, increasing the amount ofunlabeled symmetrically dimethylated arginine analyte in the sample canresult in decreased binding of the fluorescent-labeled symmetricallydimethylated arginine analyte by the symmetrically dimethylated arginineanalyte antibody, and thereby decrease the polarization of light emittedfrom sample. The quantitative relationship between polarization andconcentration of the unlabeled symmetrically dimethylated arginineanalyte in the sample can be established by measuring the polarizationvalues of calibrations with known concentrations of symmetricallydimethylated arginine analyte. Thus, FPIA can be used to identify thepresence and concentration of symmetrically dimethylated arginineanalyte in a sample.

Homogeneous microparticles immunoassay technology, which can be referredto as immunoturbidimetric assays, is based on the agglutination ofparticles and compounds in solution. When particles and/or chemicalcompounds agglutinate, particle sizes can increase and increase theturbidity of a solution. Accordingly, symmetrically dimethylatedarginine analyte antibodies can be used with microparticles in order toassess the presence, and optionally the amount, of symmetricallydimethylated arginine analyte in a sample. Homogeneous microparticlesimmunoassay may be useful because the immunoassays can be performed onblood, blood hemolysate, serum, plasma, tissue, and/or other samples.Homogeneous microparticles immunoassay assays can be configured to beperformed with symmetrically dimethylated arginine analyte and loadedonto a microparticle, or with a symmetrically dimethylated arginineanalyte antibody loaded onto a microparticle. Homogeneous microparticlesimmunoassay or immunoturbidimetric assays find use for measuringagglutination of substances in a sample. Immunoturbidimetric assaytechnologies are described in, e.g., U.S. Pat. Nos. 5,571,728,4,847,209, 6,514,770, and 6,248,597, the disclosures of which areincorporated herein by reference. Such assays involve light attenuation,nephelometric, or turbidimetric methods.

Cloned Enzyme Donor Immunoassays (“CEDIA®”, ThermoFisher), as are basedupon the competition of symmetrically dimethylated arginine analyte inthe biological sample with a symmetric dimethylarginine conjugatecontaining an inactive genetically engineered enzyme-donor (“ED”)fragment such as from β-D-galactoside galactohydrolase orβ-galactosidase (“β-gal”) from E. coli, for binding to an antibodycapable of binding symmetrically dimethylated arginine analyte. Ifsymmetrically dimethylated arginine analyte is present in the sample itmay bind to the antibody, leaving the ED portion of the ED-derivativeconjugate free to restore enzyme activity of β-D-galactosidegalactohydrolase or β-gal in the reaction mixture so as to be capable ofassociation with enzyme acceptor (“EA”) fragments. The active enzyme,which includes the ED and EA, may then be capable of producing aquantifiable reaction product when exposed to an appropriate substrate.An example of a substrate is chlorophenol red-β-D-galactopyranoside(“CPRG”), which can be cleaved by the active enzyme into galactose andCPR, where CPR is measured by absorbency at about wavelength 570 nm. Ifsymmetrically dimethylated arginine analyte is not present in thesample, the antibody may bind to the ED-derivative conjugate, therebyinhibiting association of the ED fragments with the EA fragments andinhibiting restoration of enzyme activity. The amount of reactionproduct and resultant absorbance change are proportional to the amountof symmetrically dimethylated arginine analyte in the sample.

A competitive assay using chemiluminescent microparticle immunoassay(“CMIA”) technology can also be used to assess whether or notsymmetrically dimethylated arginine analyte is present in a sample.Various types of CMIA technologies may be used for determining thepresence and/or amount of an analyte in a sample. CMIA assays caninclude the use of symmetrically dimethylated arginine analyteantibodies, which are capable of binding to symmetrically dimethylatedarginine analyte, which are coupled to particles, such as magneticparticles or particles suitable for separation by filtration,sedimentation, and/or other means. Additionally, a tracer, which caninclude a symmetric dimethylarginine or derivative linked to a suitablechemiluminescent moiety, can be used to compete with free symmetricallydimethylated arginine analyte in the patient's sample for the limitedamount of symmetrically dimethylated arginine analyte antibody on theparticle. After the sample, tracer, and antibody particles interact anda routine wash step has removed unbound tracer, the amount of tracerbound to antibody particles can be measured by chemiluminescence,wherein chemiluminescence is expressed in Relative Light Units (RULE).The amount of chemiluminescence is inversely related to the amount offree analyte in the patient's sample and concentration is determined byconstructing a standard curve using known values of the analyte.

According to some embodiments, provided is a homogenous enzymeimmunoassay for the analysis of SDMA in biological fluids (e.g., wholeblood, blood serum, blood plasma, urine, sputum, semen, saliva, ocularlens fluid, cerebral fluid, spinal fluid, amniotic fluid, tissue culturemedia, or the like, and including dilutions thereof). The assay is basedon competition between SDMA present in the biological fluid andNα-acylated-SDMA or Nα-alkylated-SDMA labeled with an enzyme (e.g.,glucose-6-phosphate dehydrogenase (G6PDH)) for antibody binding sites.Enzyme activity decreases upon binding to the antibody, so the SDMAconcentration in the biological fluid can be measured in terms of enzymeactivity. For example, active G6PDH converts nicotinamide adeninedinucleotide (NAD⁺) to NADH, resulting in an absorbance change that maybe measured spectrophotometrically. A bacterial (Leuconostocmesenteroides) enzyme may be employed in the assay so that endogenousserum G6PDH does not interfere because the coenzyme NAD⁺ functions onlywith the bacterial enzyme.

In certain embodiments, in an immunoassay of the present disclosure,including in any of the homogenous enzyme immunoassays described herein,one or more reagents are provided in lyophilized form. As just oneexample, a composition that includes any of the compounds of Formula 1of the present disclosure, any of the antibodies of the presentdisclosure, or both, may be provided in the form of lyophilized reagentspheres (or “beads”) as described in U.S. Pat. No. 5,413,732, thedisclosure of which is incorporated herein by reference in its entiretyfor all purposes. Briefly, such spheres may be made, e.g., by forming ahomogeneous solution of a reagent; measuring uniform drops of thesolution (e.g., 2 to 50 μL); dispensing the uniform, measured drops intoan unagitated cryogenic liquid (e.g., liquid nitrogen), whereby thedrops are frozen; collecting the frozen drops from the cryogenic liquid;and lyophilizing the frozen drops, thereby forming a plurality oflyophilized reagent spheres.

According to some embodiments, e.g., including embodiments in which oneor more reagents are provided in lyophilized form, a centrifugalanalyzer that includes a microfluidic rotor (or “disc”) is employed inan immunoassay of the present disclosure. For example, an immunoassay ofthe present disclosure may employ an analyzer that includes acentrifugal rotor for separating plasma from whole blood that includes aplurality of internal chambers and passages for combining blood plasmaor serum with one or more reagents (e.g., lyophilized spheres asdescribed above) and distributing the plasma or serum to a plurality ofindividual test wells. The chambers and passages necessary forseparating the whole blood into plasma are located radially outward frommetering chambers that deliver precisely measured volumes of bloodand/or diluent to a separation chamber. The separation chamber includesa radially-outward cell trap. Spinning of the rotor causes the cellularcomponents of the whole blood to be sequestered in the cell trap. Theseparated plasma is then delivered to a plurality of test wells orcuvettes. The above separation and aliquoting steps typically occur as aresult of centrifugal force generated by the spinning rotor. Thelyophilized reagent spheres described above in combination with therotors described above are particularly suitable for analyzing bloodplasma or diluted blood plasma. They are also useful with a wide varietyof other biological fluids, such as urine, sputum, semen, saliva, ocularlens fluid, cerebral fluid, spinal fluid, amniotic fluid, and tissueculture media, as well as food and industrial chemicals, and the like.Details regarding such centrifugal analyzers that include a microfluidicrotor may be found, e.g., in U.S. Pat. Nos. 5,061,381, 5,173,193;5,122,284 and 5,186,844, the disclosures of which are incorporatedherein in their entireties for all purposes.

In some embodiments, the immunoassay employs a centrifugal analyzer thatincludes a microfluidic rotor comprising siphons for delivering apremeasured volume of liquid (e.g., a biological sample such as wholeblood, blood serum, or blood plasma) between a first and a secondchamber in the rotor. The siphons may include an elbow that is radiallyinward of the radially most inward point of the fluid in the firstchamber. As the rotor is spinning, the fluid does not flow past theelbow. Once the rotor stops, capillary forces “prime” the siphon bypulling fluid just around the elbow. When the rotor is restarted,centrifugal force draws the remaining fluid out of the metering chamberinto the receiving chamber until the level of the fluid in the meteringchamber is at the same radial distance as the outlet of the siphon. Thesiphons may be designed such that the inlet of the siphon on the firstchamber is radially outward of the siphon outlet on the second chamber.The positioning of the inlets and outlets of the siphons providescertain advantages. For example, the inlet of the siphon may always bepositioned radially outward of the final position of the meniscus of thefluid in the first chamber, after fluid has been transferred to thesecond chamber. Thus, inaccuracy in measurement associated withdifferent shaped menisci in different fluids is minimized since themeniscus is minimized. In addition, as will be appreciated by one ofskill in the art, all siphons are semi-stable because the train of fluidin a siphon is stable but easily broken if the rotor is perturbed. Whenthe train of fluid is broken, under centrifugal force, the fluidcontained in the siphon will flow to the radially most outward point. Inprevious siphons, this point is the siphon outlet. Thus, the potentialexists for the delivery of unmetered volumes of fluid to the receivingchamber. In the siphons described herein, the radially most outwardpoint in the siphon is the siphon inlet. In this design, the problem ofdelivering unmetered volumes of fluid is avoided because the fluid flowsback into the first chamber when the train of fluid is broken. Furtherdetails regarding analyzers that include a centrifugal rotor comprisingsiphons for delivering premeasured volumes of liquid, which may beemployed in any of the methods/immunoassays of the present disclosure,may be found in U.S. Pat. No. 7,998,411, the disclosure of which isincorporated herein by reference in its entirety for all purposes.

The symmetric dimethylarginine derivatives, conjugates, antibodies,immunogens, and/or other conjugates described herein are also suitablefor any of a number of other heterogeneous immunoassays with a range ofdetection systems including but not limited to enzymatic or fluorescent,and/or homogeneous immunoassays including but not limited to rapidlateral flow assays, and antibody arrays, as well as formats yet to bedeveloped.

While various immunodiagnostic assays have been described herein thatutilize the symmetric dimethylarginine derivatives, conjugates,antibodies, and immunogens, such assays can also be modified. As such,various modifications of steps or acts for performing such immunoassayscan be made within the scope of the embodiments described herein.Additional information related to assay format are described, amongother places, in David Wild (The Immunoassay Handbook, 4th EditionPublished Date: 31 Jan. 2013, Elsevier Science).

Kits

Aspects of the present disclosure further include kits. In someembodiments, the kits find use in determining an amount of at least onesymmetrically dimethylated arginine analyte in a sample. Such kits mayinclude any of the compounds of Formula 1 of the present disclosure, anyof the antibodies of the present disclosure, or both.

In certain embodiments, provided are kits for determining an amount ofat least one symmetrically dimethylated arginine analyte in a sample,such kits including any of the antibodies of the present disclosure, andinstructions for using the antibody to determine an amount of at leastone symmetrically dimethylated arginine analyte in a sample. Accordingto some embodiments, the antibody specifically binds to a metabolite ofsymmetric dimethylarginine (non-limiting examples of which includesymmetric Nα-acetyl-dimethylarginine (Ac-SDMA)), and the kit may furtherinclude instructions for determining an amount of the metabolite ofsymmetric dimethylarginine. In certain embodiments, the kits thatinclude an antibody of the present disclosure further include any of thecompounds of Formula 1 of the present disclosure. In some embodiments,the compound is one where Z is a label, such as an enzyme, e.g.,glucose-6-phosphate dehydrogenase (G6PDH).

Also provided are kits for determining an amount of at least onesymmetrically dimethylated arginine analyte in a sample, the kitsincluding any of the compounds of Formula 1 of the present disclosure,and instructions for using the compound to determine an amount of atleast one symmetrically dimethylated arginine analyte in a sample. Incertain aspects, the compound is one where Z is a label, such as anenzyme, e.g., glucose-6-phosphate dehydrogenase (G6PDH). Such kits mayfurther include any of the antibodies of the present disclosure.According to some embodiments, the antibody specifically binds to ametabolite of symmetric dimethylarginine (non-limiting examples of whichinclude symmetric Nα-acetyl-dimethylarginine (Ac-SDMA)), and the kit mayfurther include instructions for determining an amount of the metaboliteof symmetric dimethylarginine.

According to some embodiments, the kits of the present disclosure areuseful for conveniently performing an assay for the determination of asymmetrically dimethylated arginine analyte in a sample, such as, forexample, symmetric dimethylarginine and its analog, symmetricNα-acetyl-dimethylarginine. The kit may include: (a) an antibody raisedthat specifically binds to symmetric dimethylarginine and a compound ofFormula 1 described herein (e.g., a symmetric dimethylarginineconjugate); and (b) instructions for determining the amount of thesymmetrically dimethylated arginine analyte in the sample. In someembodiments, the kit also includes a conjugate of a compound of Formula1, where the conjugate includes a label (e.g., a detectable label, suchas G6PDH). In certain instances, the kit also includes ancillaryreagents for determining the analyte. The antibody of the kit may be anantibody raised against a compound of Formula 1 described herein.

To enhance the versatility of the immunoassay, the kit reagents can beprovided in packaged combination, in the same or separate containers, inliquid or lyophilized form so that the ratio of the reagents providesfor substantial optimization of the method and assay. In certainembodiments, the kits include an antibody of the present disclosure, acompound of the present disclosure, or both, provided as lyophilizedreagent spheres as described in U.S. Pat. No. 5,413,732, the disclosureof which is incorporated herein by reference in its entirety for allpurposes. The reagents provided in the kits may each be in separatecontainers or various reagents can be combined in one or morecontainers, e.g., depending on the cross-reactivity and stability of thereagents. In some embodiments, the compound of Formula 1 describedherein (e.g., a symmetric dimethylarginine conjugate) is present inlyophilized form. In some embodiments, the antibody is present inlyophilized form. For example, the compound of Formula 1 can be presentin a first lyophilized composition (which may further include one ormore excipients, buffers, stabilizers, etc.), and the antibody can bepresent in a second lyophilized composition (which may further includean enzyme substrate and one or more excipients, buffers, stabilizers,etc.). The first lyophilized composition and the second lyophilizedcomposition may be provided in a single kit, such as for example in apackaging or container for single use.

The kit can further include other separately packaged reagents forconducting an assay such as ancillary reagents such as an ancillaryenzyme substrate, and so forth. The relative amounts of the variousreagents in the kits can be varied widely to provide for concentrationsof the reagents that substantially optimize the reactions that need tooccur when performing a method/immunoassay (e.g., homogenous enzymeimmunoassay) and further to optimize substantially the sensitivity ofthe assay. Under appropriate circumstances one or more of the reagentsin the kit can be provided as a dry powder, usually lyophilized,including excipients, which on dissolution will provide for a reagentsolution having the appropriate concentrations for performing a methodor assay in accordance with the present invention. The kit can furtherinclude a written description of a method in accordance with the presentinvention as described above.

The description of certain exemplary embodiments of kits uses thelanguage “and/or,” which means that the kit may or may not contain eachitem mentioned. This language is used for the sake of brevity. Ingeneral, an immunoassay kit will include at least one antibody for animmunogen of an analyte, e.g., symmetric dimethylarginine, and at leastone enzyme conjugate (e.g., label conjugate) that corresponds to thatanalyte, e.g., an enzyme conjugate of a derivative of symmetricdimethylarginine.

In certain embodiments, a kit is provided for an assay for the analytesymmetric dimethylarginine and/or metabolites of symmetricdimethylarginine. The kit may include, in packaged combination: (i) anantibody raised against a compound of Formula 1; and (ii) a conjugate ofa derivative of the analyte. Another embodiment of the presentlydisclosure is a kit for an assay for the analyte symmetricdimethylarginine and/or metabolites of symmetric dimethylarginine thatincludes, in packaged combination: (i) an antibody raised against aderivative of the analyte; and (ii) a conjugate of a hapten of theanalyte, where the hapten is a compound of Formula 1.

The compounds, methods and kits of the present disclosure find use inroutine monitoring of symmetrical dimethylarginine by immunoassays. Incertain embodiments, these immunoassays provide simple automated testsadapted to standard laboratory equipment with a quick turn-around time.As described herein, in order to provide such immunoassays, antibodiesto a symmetrical dimethylated arginine analyte are produced. Thederivatives and immunogens are designed to impart, through thecorresponding antibodies, specific reactivity to symmetricaldimethylarginine.

The instructions included in the kits may be recorded on a suitablerecording medium. For example, the instructions may be printed on asubstrate, such as paper or plastic, etc. As such, the instructions maybe present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or sub-packaging) etc. In other embodiments, the instructionsare present as an electronic storage data file present on a suitablecomputer readable storage medium, e.g., portable flash drive, DVD,CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, the means for obtaining theinstructions is recorded on a suitable substrate.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. By “average” is meant the arithmeticmean. Standard abbreviations may be used, e.g., bp, base pair(s); kb,kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h orhr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt,nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,subcutaneous(ly); and the like.

In relation to the compounds and conjugates and immunogens, thefollowing abbreviations are used: DCM is dichloromethane; DMF isN,N-dimethylformamide; EDTA is ethylenediaminetetraaceticacid; KLH iskeyhole limpet hemocyanin; SATA is N-succinimidyl S-acetylthioacetate;TFA is trifluoroacetic acid; EDCI is1-ethyl-3(3-dimethylaminopropyl)carbodiimidehydrochloride; NHS isN-hydroxysuccinimide; DTT is dithioerythritol; G6PDH isGlucose-6-Phosphate Dehydrogenase; EtOAc is Ethyl acetate; BSA is bovineserum albumin; DMO is Dess-Martin periodinane; MeCn is Acetonitrile; EAis ethyl acetate; t-Boc is tert-butyloxycarbonyl protecting group; TLCis thin layer chromatography; MeOH is methanol; AcOH is acetic acid;PBST is phosphate buffered saline with Tween-20; TMB is3,3′,5,5′-tetramethylbenzidine; PBMC is peripheral blood mononuclearcell.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any purificationprotocol known in the art, including chromatography, such as HPLC,preparative thin layer chromatography, flash column chromatography andion exchange chromatography. Any suitable stationary phase can be used,including normal and reversed phases as well as ionic resins. In certainembodiments, the disclosed compounds are purified via silica gel and/oralumina chromatography. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, New York, 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas J. F. W. McOmie, “Protective Groups in Organic Chemistry”, PlenumPress, London and New York 1973, in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer),Academic Press, London and New York 1981, in “Methoden der organischenChemie”, Houben-Weyl, 4th edition, Vol. 15/1, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

The subject compounds can be synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods. A varietyof examples of synthetic routes that can be used to synthesize thecompounds disclosed herein are described in the schemes below.

Example 1 Preparation of SDMA-M Hapten

Step 1.

To a solution of (1) (534 mg, 6.0 mmol, 1.0 equiv.) in saturated NaHCO₃(12 mL) was added (2) (930 g, 6.0 mmol, 1.0 equiv.) in small portionsmaintaining the temperature at 0° C. Upon completion of addition, theresulting solution was stirred at 0° C. for 1.5 hours. LC-MS showed allstarting material was converted to target material. The stirred reactionmixture was allowed to warm to room temperature and extracted withEtOAc. The crude product was purified on silica gel columnchromatography (eluting with Petroleum ether/Ethyl acetate=1/1, v/v) togive (3) (0.91 g, 91% yield) as clear oil. The structure was confirmedby H NMR.

Step 2.

A mixture of (3) (0.9 g, 5.3 mmol, 1.0 equiv.), Dess-Martin periodinanereagent (4.5 g, 10.6 mmol, 2.0 equiv.) and NaHCO₃ (7.9 g, 94.3 mmol, 18equiv.) in DCM (40 mL) was stirred at 25° C. for 3 hours. TLC showedmost starting material transformed to target material. The reactionmixture was filtered and the resulting filtrate was concentrated undervacuum. The crude product was purified by silica gel columnchromatography (Petroleum Ether/Ethyl Acetate=1/1, v/v) to give 0330-04(0.35 g, 40% yield) as clear oil. The product (4) was extremelysensitive to air, and was immediately used for the next step. Thestructure was confirmed by LC-MS.

Step 3.

To a solution of (4) (668 mg, 4 mmol, 2.0 equiv.) and symmetricdimethylarginine (SDMA) (404 mg, 2 mmol, 1.0 equiv.) in MeOH was addedAcOH (200 μL) and NaBH₃CN (150 mg, 2.4 mmol, 1.2 equiv.). The resultingmixture was stirred at room temperature for 3 hours. LC-MS showed moststarting material transformed to target material. The reaction wasquenched with water and immediately purified by Biotage reverse phasechromatography to give SDMA-M (326 mg, 35% yield, as its TFA salt) asclear oil. The structure was confirmed by ¹H NMR. H NMR (400 MHz, D₂O):δ 6.81 (s, 2H), 3.97 (M, 1H), 3.51 (m, 2H), 3.21 (m, 2H), 3.08 (m, 2H),2.78 (s, 6H), 1.98 (m, 2H), 1.64 (m, 6H). (see FIG. 5).

Example 2 Preparation SDMA-SBAP Hapten

Step 1.

To a solution of (5) (1.4 g, 7.2 mmol, 1.0 equiv.), andN-hydroxysuccinimide (NHS) (1 g, 8.7 mmol, 1.2 equiv.) in DMF (18 mL)was added EDCI (2.78 g, 14.5 mmol, 2.0 equiv.). The resulting mixturewas stirred at RT for 12 hours. LC-MS showed all the starting materialtransformed to target material. The solution was poured into H₂O (100mL) and extracted with EA (100 mL). The organic phase was washed withH₂O and saturated brine. The organic solvent was removed under reducedpressure and dried in vacuum to give desired product (6) (2.0 g 95.2%yield). The structure was confirmed by ¹H NMR.

Step 2.

To the solution of symmetric dimethylarginine (SDMA) (400 mg, 2.0 mmol,1.0 equiv.) in DMF (5 mL) was added (6) (630 mg, 2.2 mmol, 1.1 equiv.).The reaction mixture was stirred at 60° C. for 12 hours. LC-MS showedall the starting material transformed to target material. Solvent wasremoved under vacuum. The crude product was dissolved in MeCN and water(7.5 mL, 1/1, v/v) and then purified using Biotage reverse phasechromatography (standard method) to give the pure product (7) (650 mg,salt of TFA, 66.7% yield) as a clear oil. The structure was confirmed by¹H NMR.

Step 3.

To the solution of (7) (650 mg, 1.33 mmol, 1.0 equiv.) in MeCN (10 mL)was added TFA (4 mL). The reaction mixture was stirred at RT for 30 min.LC-MS showed all the starting material transformed to target material(8). Solvent was removed under reduced pressure and dried in vacuum togive desired product in quantitative yield (as a TFA salt). Thestructure was confirmed by LC-MS.

Step 4.

A mixture of (8) (258 mg, 0.87 mmol, 1.0 equiv.), N-Succinimidylbromoacetate (9) (205 mg, 0.87 mmol, 1.0 equiv.) and DIPEA (0.3 mL, 1.74mmol, 2.0 equiv.) in DMF (2 mL) was stirred at 30° C. for 12 hours.LC-MS showed all the starting material transformed to target material.Solvent was removed under vacuum. The crude product was dissolved inMeCN and water (3.5 mL, 1/1, v/v) and then purified with Biotage reversephase chromatography (standard method) to give the pure productSDMA-SBAP (175 mg, salt of TFA, 39.6% yield) as a clear oil. Thestructure was confirmed by ¹H NMR. ¹H NMR (400 MHz, D₂O) δ 4.36-4.32 (m,1H), 3.83 (s, 2H), 3.45 (t, J=6.4, 2H), 3.18-3.15 (m, 2H), 2.76 (s, 6H),2.49 (t, J=6.4, 2H), 1.87-1.74 (m, 1H), 1.73-1.58 (m, 3H). The reactionscheme is shown in FIG. 6.

Example 3 Preparation of SDMA-M-SH-KLH Immunogen

Hapten SDMA-M was conjugated to KLH where thiol groups were chemicallyintroduced to KLH. N-Succinimidyl-S-acetylthioacetate was reacted withthe primary amines of KLH, which added protected sulfhydryls.Deprotection of protected sulfhydryls with hydroxyl amine produced thedesired thiolated SH-KLH (FIG. 8). Conjugation of hapten SDMA-M withSH-KLH resulted in immunogen SDMA-M-SH-KLH (see FIG. 8).

a) Preparation of SH-KLH

Lyophilized KLH (20 mg) was reconstituted with deionized water and pHadjusted to 8.6 with 1 M carbonate-bicarbonate buffer. A solution ofN-Succinimidyl S-acetylthioacetate was prepared (4.67 mg was dissolvedin 92 μL of DMF to a concentration of 220 mM) and slowly added to theKLH solution over 4 hrs. The reaction mixture was stirred at roomtemperature while N-Succinimidyl S-acetylthioacetate was being added,then stirred in the cold-room (4° C.) for an additional 16 hours.

Deacylation to generate a sulfhydryl for use in cross-linking wasaccomplished by adding 200 μL of a deacetylation solution (0.7 Mhydroxylamine solution in 12.5 mM NaH₂PO₄—Na₂HPO₄ buffer, pH 7).Contents were mixed and reaction incubated for 2 hours at roomtemperature resulting in the product SH-KLH (see FIG. 8). EDTA was addedat the end of this reaction to a concentration of 1 mM.

b) Preparation of SDMA-M-SH-KLH Immunogen

Dithiothreitol solution was added to the above SH-KLH solution for atotal concentration of 1 mM to minimize disulfide bond formation. The pHwas adjusted to 7.2 with 1 M carbonate-bicarbonate buffer. To the SH-KLHsolution, 146 μl of maleimide derivative hapten SDMA-M dissolved in DMF(9.6 mg dissolved in 0.2 mL DMF) was added slowly over 4 to 5 hrs. Thereaction was continued overnight at 4° C. The mixture was purified bydialysis using a 10,000 MWCO Slide-A-Lyzer® Dialysis Cassette (Pierce)in 4 liter NaH₂PO₄—Na₂HPO₄ buffer 12.5 mM, pH 7.0, at 2-8° C. Thisprocedure yielded immunogen SDMA-M-SH-KLH (see FIG. 8).

Example 4 Preparation of SDMA-SBAP-SH-G6PDH Conjugate

Hapten SDMA-SBAP prepared as described in Example 2 is designed forproteins containing cysteine groups such as mutant G6PDH (see U.S. Pat.Nos. 6,455,288, 6,090,567, 6,033,890) or introduction of thiol-groups toG6PDH similarly as described in Example 3 by chemical reactions.

SDMA-SBAP hapten (7 mg, 0.018 mmol) was dissolved in DMF (0.21 mL). Thesolution was stirred at room temperature for 30 minutes. This SDMA-SBAPsolution was used as described below.

Dithiothreitol solution was added to mutant G6PDH at a concentration of2 mM to reduce cysteine thiol groups connected in disulfide bonds tosulfhydryl groups. The resulting enzyme solution (0.9 mg, 1.5 mL) wasadjusted to pH 7.2 with 1 M carbonate bi-carbonate buffer and mixed withapproximately 340 fold molar excess (0.07 mL) of SDMA-SBAP hapten. Thereaction mixture was allowed to stir gently at 4° C. for 16 hours.Excess SDMA-SBAP hapten was separated from the enzyme-hapten conjugateby passing the reaction mixture over a column of Sephadex G-50 in 12.5mM NaH₂PO₄—Na₂HPO₄ buffer, pH 7.0. The column fractions containing theenzyme-hapten conjugate are pooled by measuring absorption at 280 nm togive conjugate SDMA-SBAP-SH-G6PDH (see FIG. 7).

Hapten SDMA-SBAP was conjugated with BSA using a conjugation proceduresimilar to that described above. The SDMA-SBAP-SH-BSA conjugate (FIG. 9)was used to screen B-cells and monoclonal antibodies by indirect ELISAas described in Example 6.

Example 5 Preparation of Polyclonal Antibodies to SDMA

Twenty-four female white New Zealand rabbits were immunized by injectingsubcutaneously 200 μg/rabbit of SDMA-M-SH-KLH immunogen, as prepared inExample 3, emulsified in Complete Freund's adjuvant. The rabbits wereboosted every four weeks after the initial injection with 100 μg/rabbitof the same immunogen emulsified in Incomplete Freund's Adjuvant. Onehundred and thirty-four days after the initial immunization, bleedscontaining polyclonal antibodies from each rabbit were obtained from thecentral ear artery. The anti-serum from these bleeds containingSDMA-M-SH-KLH antibodies were evaluated in a homogeneous assay format(FIG. 10) by measuring maximum antibody inhibition of enzyme conjugateSDMA-SBAP-SH-G6PDH and modulation in the presence of symmetricdimethylarginine as described in Examples 7 and 8.

From these experiments rabbits 21342 and 26494 were selected to isolatePBMC's as a source of B-cells for cloning (Example 6) from rabbitsimmunized with SDMA-M-SH-KLH immunogen. Initial screening results of the24 rabbit polyclonal antisera immunized with SDMA-M-SH-KLH is shown inTable 1 below.

TABLE 1 Max. Max. Inhibition Modulation Inhibition Modulation Rabbit ID(%) (%) Rabbit ID (%) (%) #21331 32 8 #26490 23 8 #21332 39 10 #26491 42 #21333 17 6 #26492 26 6 #21334 39 12 #26493 11 2 #21335 32 12 #2649436 22 #21336 31 7 #26496 31 9 #21337 27 4 #26497 14 3 #21338 52 13#26498 4 0 #21339 51 13 #26499 6 4 #21340 11 2 #26500 31 5 #21341 32 8#26501 25 8 #21342 48 14 #27410 45 16

Example 6 Preparation Rabbit Monoclonal Antibody

Rabbit recombinant monoclonal antibodies were prepared using singleB-cell screening strategy for efficiently sampling the natural antibodyrepertoire of immunized rabbits. This technique is generally applicableto produce monoclonal antibodies to symmetric dimethylarginine asdescribed herein.

Rabbit Peripheral blood mononuclear cells (PBMC's) from immunizedanimals #21342 and #26494 were used as a source of B-cells. PBMC's wereisolated from approximately 40 mL whole blood from each rabbit usingstandard density gradient centrifugation procedure. The PBMC's werediluted in PBS and theoretically dispensing single cell per well intoforty 96-well plates the same day and cultured.

Resulting supernatants from each well were tested by indirect ELISAagainst SDMA-SBAP-SH-BSA antigen (FIG. 9). Forty 96-well microtiterplates were coated with 0.1 μg/well SDMA-SBAP-SH-BSA in 0.1 M carbonatebuffer, pH 9.5 and stored over night at 4° C. The plates were emptiedand then blocked with 3% skimmed milk powder in PBST with shaking for 1hr at RT. After flicking off the blocking solution the plates wererinsed with PBST. Twenty-five μL of PBST were added to each wellfollowed by adding 25 μL of cell supernatant to all the wells andincubated for 1 hr at 37° C. in an incubator. Plates were washed 5× withPBST with a total wash time of 30 min. Afterwards, 100 μL/well secondaryantibody 1:10,000 (v/v) goat anti-rabbit IgGFc-HRP conjugate in PBST wasadded and plates were incubated for 1 hour at 37° C. under constantshaking. Plates were washed 5 times with PBST, total wash time of 30min. TMB substrate was added at 50 uL/well, plates. After a five minuteincubation in the dark for color to develop, the reaction was stopped bythe addition of 50 μL of 1 N HCl. Color was read via a microplate readerat 450 nm, and data was transferred to a computer for analysis.Supernatants that bound the SDMA-M-SH-BSA conjugate (produced color inthe wells) were considered positives. A total of 32 cells were selectedfor cloning and expression.

For each ELISA well with an antigen-specific antibody, mRNA was isolatedfrom the corresponding PBMC culture well and divided to separatelysynthesize cDNA from the variable regions of the rabbit genes, IgH andIgK. After two rounds of PCR to amplify, the cDNA was seamlessly ligatedinto separate mammalian expression vectors with a constant IgG region ofthe heavy chain or the constant IgK region of the light chain,respectively. Ligation mixtures were transformed into E. coli to selectcorrect expression constructs and cultured to isolate plasmid. Theexpression constructs were co-transfected into HEK293 cells. Transfectedcells were cultured 2 days to secrete the recombinant antibody. Theantigen binding property of the recombinantly expressed antibodies wereassessed by indirect ELIA against SDMA-SBAP-BSA antigen as describedabove. The clones selected were further used for evaluation in thehomogeneous enzyme immunoassay format as described on Examples 7 and 8.DNA sequencing was performed for selected rabbit monoclonal antibodiesand translated with a standard code to provide protein sequence data forall heavy chain and kappa chain variable region sequences. The Tablebelow summarizes the development phases resulting in monoclonalantibodies.

TABLE 2 Enrichment Cloning & & Selection Expression Immunization PhasePhase Phase No. B cells positive ID of clones Rabbit ID by indirectELISA selected for No. rabbits no. selected against SDMA- No. of cellscloned testing immunized Immunogen for cloning SBAP-BSA antigen andexpressed and sequencing 24 KLH-SH- #21342 190 23 1H4/1K4 SDMA-M 8H1/8K318H1/18K2 #26494  58 29 1H2/1K4 3H1/3K3 5H1/5K1

Example 7

Preparation of SDMA Standards and Calibration Protocol Symmetricdimethylarginine di(p-hydroxyazobenzene-p′-sulfonate) salt (Sigma) wasdissolved in DMF:H₂O (1:1), and diluted in a synthetic matrix to a finalconcentration of SDMA of 4 g/ml stock solution. SDMA stock solution wastransferred into aliquots of synthetic matrix to achieve levels of 0,15, 50, 100, 150, 200 μg/dL. The synthetic matrix consisted of aproteinaceous solution buffered with HEPES (6.5 mM, pH 6.7), and EDTA(0.25 mM), surfactant, defoaming agent and a preservative. The standardswere validated by LC/MS (see FIG. 17).

A six-point calibration curve was used to quantify symmetricallydimethylated arginine in samples. Calibration curves were generated onthe Beckman Coulter AU480 automated clinical chemistry analyzer (seeExamples 7 and 8) by measuring each calibrator level in duplicate.

Nα-acetyl-dimethylarginine (Ac-SDMA) calibrators were prepared in asimilar manner as described above. Nα-acetyl-dimethylarginine wassynthesized by SYNthesis med chem (Australia).

Example 8 Reagents and Assays

Symmetric dimethylarginine antibodies and enzyme conjugates may beadvantageously used in accordance with the present disclosure in ahomogeneous assay format to detect a symmetric dimethylated arginineanalyte in samples. Antibodies may be evaluated by known methods, suchas, conjugate inhibition, conjugate modulation, calibration,cross-reactivity and spike-recovery. For these purposes, antibody(polyclonal antibodies rabbit #21342, rabbit #26494, or rabbit #27420,or cloned antibodies 1H4/1K4, 8H1/8K3, 18H1/18K2, 1H2/1K4, 3H1/3K3, or5H1/5K1) is added into the antibody diluent to prepare the antibodyreagent. The antibody reagent includes antibody as prepared above,buffer, salts, stabilizers, preservatives, NAD⁺, andglucose-6-phosphate. Enzyme conjugate SDMA-SBAP-SH-G6PDH is added intothe conjugate diluent to prepare the enzyme conjugate reagent. Theenzyme conjugate reagent includes the conjugate, buffer, stabilizers,salts, and preservatives.

A clinical chemistry analyzer useful to evaluate antibodies and enzymeconjugates in a homogeneous enzyme immunoassay format is the BeckmanCoulter AU480 (Beckman Coulter, Brea, Calif.). The Beckman AU480 is anautomated biochemistry spectrophotometer analyzer used by medicallaboratories to process biological fluid specimens, such as urine,cerebrospinal fluid, oral fluids, plasma and serum. The analyzer iscapable of maintaining a constant temperature, pipetting samples, mixingreagents, measuring light absorbance and timing the reaction accurately.

A homogeneous enzyme immunoassay is conducted using a liquid,ready-to-use, two reagent assay as described above. Typically, 2-15 μLsymmetrically dimethylated arginine analyte-containing sample isincubated with 75-150 μL antibody reagent followed by the addition ofthe 50-100 μL enzyme conjugate reagent.

The assay is a homogeneous enzyme immunoassay technique used for theanalysis of SDMA in biological fluids. The assay is based on competitionbetween SDMA in the specimen and Nα-acylated-SDMA or Nα-alkylated-SDMAlabeled with the enzyme glucose-6-phosphate dehydrogenase (G6PDH) forantibody binding sites. Enzyme activity decreases upon binding to theantibody, so the drug concentration in the sample can be measured interms of enzyme activity. Active enzyme converts nicotinamide adeninedinucleotide (NAD⁺) to NADH, resulting in an absorbance change that ismeasured spectrophotometrically at 340 nm. Endogenous serum G6PDH doesnot interfere because the coenzyme NAD⁺ functions only with thebacterial (Leuconostoc mesenteroides) enzyme employed in the assay. Thechange in the absorbance at 340 nm can be measuredspectrophotometrically and is proportional to the enzyme conjugateactivity which in turn is related to analyte concentration (see FIG.10).

Example 9 Antibodies and Calibration Using Symmetric DimethylarginineAnalyte

Symmetric dimethylated arginine analyte antibodies and enzyme conjugate(SDMA-SBAP-SH-G6PDH) were used in a homogeneous assay format to generatecalibration curves using symmetric dimethylarginine standards asdescribed in Example 7. Antibody reagents were prepared as described inExample 8 using symmetric dimethylated arginine analyte polyclonalantibodies rabbit #21342, rabbit #26494, rabbit #27420 and clonedantibodies 1H4/1K4, 8H1/8K3, 18H1/18K2, 1H2/1K4, 3H1/3K3, or 5H1/5K1.Enzyme conjugate SDMA-SBAP-SH-G6PDH was used to prepare the conjugatereagent. A 6-point calibration curve was generated on the Beckman AU480clinical chemistry analyzer as described in Examples 7 and 8. Typicalcalibration curves are shown in the table below and dose-response curvesshown in FIG. 11 and FIG. 12. These experiments demonstrated thatpolyclonal antibodies rabbit #21342, rabbit #26494, rabbit #27420 andcloned monoclonal antibodies 1H4/1K4, 8H1/8K3, 18H1/18K2, 1H2/1K4,3H1/3K3, or 5H1/5K1 have antibody binding reaction to symmetricdimethylarginine demonstrating a dose-response relationship.

TABLE 3 Rabbit Polyclonal Antibody SDMA Conc. Reaction Rate (mA/min)Average of Duplicates (μg/dL) Rabbit # 21342 Rabbit # 26494 Rabbit #27420 0 528 585 576 15 559 634 624 50 607 703 690 100 656 755 734 150685 783 753 200 705 798 765 Rabbit Recombinant Antibody SDMA Conc.Reaction Rate (mA/min) Average of Duplicates (μg/dL) 1H4/1K4 8H1/8K318H1/18K2 0 535 497 492 15 564 534 531 50 617 598 595 100 669 652 649150 698 686 681 200 714 708 702 Rabbit Recombinant Antibody SDMA Conc.Reaction Rate (mA/min) Average of Duplicates (μg/dL) 1H2/1K4 3H1/3K35H1/5K1 0 592 523 574 15 630 595 617 50 687 704 671 100 736 767 710 150756 794 730 200 767 807 743

Example 10 Antibodies and Calibration Using N-Acetyl-SymmetricDimethylarginine Analyte

Symmetric dimethylated arginine analyte antibodies and enzyme conjugate(SDMA-SBAP-SH-G6PDH) were used in a homogeneous assay format to generatecalibration curves using standards prepared using Nα-Acetyl-symmetricdimethylarginine as described in Example 7. Antibody reagents wereprepared as described in Example 8 using symmetric dimethylated arginineanalyte polyclonal antibodies rabbit #21342, rabbit #26494, rabbit#27420 and cloned antibodies 1H4/1K4, 8H1/8K3, 181H1/18K2, 1H2/1K4,3H1/3K3, or 5H1/5K1. Enzyme conjugate SDMA-SBAP-SH-G6PDH was used toprepare the conjugate reagent. A6-point calibration curve was generatedon the Beckman AU480 clinical chemistry analyzer as described in Example8. Typical calibration curves are shown in the table below anddose-response curves shown in FIG. 13 and FIG. 14. These experimentsdemonstrated that polyclonal antibodies rabbit #21342, rabbit #26494,rabbit #27420 and cloned monoclonal antibodies 1H4/1K4, 8H1/8K3,181H1/18K2, 1H2/1K4, 3H1/3K3, or 5H1/5K1 have an antibody bindingreaction to Nα-Acetyl-symmetric dimethylarginine demonstrating adose-response relationship.

TABLE 4 Rabbit Polyclonal Antibody Ac-SDMA Conc. Reaction Rate (mA/min)Average of Duplicates (μg/dL) Rabbit # 21342 Rabbit # 26494 Rabbit #27420 0 519 542 571 15 533 551 576 50 559 561 578 100 591 581 589 150612 595 598 200 632 608 605 Rabbit Recombinant Antibody Ac-SDMA Conc.Reaction Rate (mA/min) Average of Duplicates (μg/dL) 1H4/1K4 8H1/8K318H1/18K2 0 521 481 480 15 538 501 498 50 569 537 535 100 603 577 575150 628 605 601 200 647 626 622 Rabbit Recombinant Antibody Ac-SDMAConc. Reaction Rate (mA/min) Average of Duplicates (μg/dL) 1H2/1K43H1/3K3 5H1/5K1 0 589 483 572 15 592 488 578 50 594 502 587 100 606 519602 150 612 529 611 200 620 546 623

Example 11 Spike-Recovery

Known amounts of symmetrically methylated arginine analyte stocksolution (4 μg/mL) were added (spike) into synthetic matrix as describedin Example 7 to achieve concentrations of 0, 15, 50, 100, 150, 200μg/dL. These samples were quantified in triplicate by the homogeneousenzyme immunoassay to confirm concentration (recovery) of the spikedsamples on the Beckman AU480 as described in Example 8. The samples werequantified using a separately prepared set of standards by generating a6-point calibration curve. Calibration curve was generated usingstandards prepared using the same analyte as the analyte beingquantified in the sample being tested. The Enzyme Conjugate Reagentcontained conjugate SDMA-SBAP-SH-G6PDH and the Antibody Reagents wereprepared containing polyclonal antibody from rabbit #21342, rabbit#26494, rabbit #27420 and antibody clones 1H4/1K4, 8H1/8K3, 18H1/18K2,1H2/1K4, 3H1/3K3, or 5H1/5K1. The symmetrically methylated arginineanalyte concentration recovered in the spike-recovery experiments werecompared to the known concentration and plotted (FIGS. 15-18). Theseexperiments demonstrated that polyclonal antibodies rabbit #21342,rabbit #26494, rabbit #27420 and cloned antibodies 1H4/1K4, 8H1/8K3,18H1/18K2, 1H2/1K4, 3H1/3K3, or 5H1/5K1 have antibody reaction tosymmetric dimethylarginine and Nα-acetyl-SDMA.

In another experiment, the same samples as above were quantified intriplicate by the homogeneous enzyme immunoassay (see Example 8) and byLC-MS-MS in duplicate. Deuterated asymmetric dimethylarginine aninternal standard was used for the LC-MS-MS method. The homogeneousenzyme immunoassay was prepared using clone 3H1/3K3 in the antibodyreagent and enzyme conjugate SDMA-SBAP-SH-G6PDH in the enzyme conjugatereagent. Results show that the homogeneous enzyme immunoassay quantifiesSDMA levels in agreement with LC-MS-MS.

In another experiment, known amounts of SDMA-Gly-Gly dipeptide wereanalyzed in the homogeneous enzyme immunoassay to confirm concentration(recovery) of the spiked samples on the Beckman AU480 as described inExample 8. Calibration curve was generated using standards preparedusing the same analyte as the analyte being quantified in the samplebeing tested. The Enzyme Conjugate Reagent contained conjugateSDMA-SBAP-SH-G6PDH and the Antibody Reagents were prepared containingantibody clone 8H1/8K3 or 3H1/3K3. The SDMA-Gy-Gly dipeptide analyteconcentration recovered in the spike-recovery experiments were comparedto the known concentration and plotted (FIG. 19).

Example 12 Antibody and Calibration using Symmetric Dimethylarginine andNα-Acetyl-Symmetric Dimethylarginine Analytes

Polyclonal antibody from rabbit #27410 and SDMA-SBAP-SH-G6PDH were usedto prepare reagents and used in homogeneous assay format to detectsymmetric dimethylated arginine and Nα-Ac-SDMA analytes as described inExample 8. The SDMA and Nα-Ac-SDMA samples were prepared as described inExample 7 and were quantified from a calibration curve generated fromstandards prepared using SDMA. Spike-recovery experiments were performedas described in Example 11 using the polyclonal antibody from rabbit#27410. FIG. 20 shows that polyclonal antibody from rabbit #27410substantially binds both SDMA and Nα-Ac-SDMA. The maximum inhibition (%)and modulation (%) for the polyclonal antibody from rabbit #27410 isshown in Table 1 above.

The preceding merely illustrates the principles of the embodiments ofthe present disclosure. It will be appreciated that those skilled in theart will be able to devise various arrangements which, although notexplicitly described or shown herein, embody the principles of theembodiments and are included within its spirit and scope. Furthermore,all examples and conditional language recited herein are principallyintended to aid the reader in understanding the principles of theembodiments and the concepts contributed by the inventors to furtheringthe art, and are to be construed as being without limitation to suchspecifically recited examples and conditions. Moreover, all statementsherein reciting principles, aspects, and embodiments of the presentdisclosure as well as specific examples thereof, are intended toencompass both structural and functional equivalents thereof.Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure.

1.-50. (canceled)
 51. A method for determining an amount of at least onesymmetrically dimethylated arginine analyte in a medium, the methodcomprising: combining in a medium: a sample suspected of containing atleast one symmetrically dimethylated arginine analyte, and an antibodythat specifically binds to a compound of Formula 1:

wherein: R¹ is —Y—Z; Y is a linking group; and Z is a label enzyme; anddetermining the presence or absence of a complex comprising thesymmetrically dimethylated arginine analyte and the antibody, whereinthe presence of the complex indicates the presence of the symmetricallydimethylated arginine analyte in the sample.
 52. The method according toclaim 51, wherein the medium further comprises the compound ofFormula
 1. 53.-55. (canceled)
 56. The method according to claim 51,wherein the label enzyme is glucose-6-phosphate dehydrogenase (G6PDH).57. The method according to claim 51, wherein the determining comprisesdetecting the presence of an enzymatic reaction product of the compound.58.-79. (canceled)
 80. The method of claim 51, wherein the linking groupcomprises 1 to 15 carbon atoms and/or 0 to 6 heteroatoms.
 81. The methodof claim 51, wherein the linking group is selected from the groupconsisting of —(CH₂)_(n)C(O)—, —C(O)(CH₂)_(n)—, —C(O)(CH₂)_(n)NHC(O)—,—C(O)(CH₂)_(n)NHC(O)(CH₂)—, —(CH₂)_(n)SCH₂C(O)—,—(CH₂)_(n)C(O)NH(CH₂)_(n)—, —(CH₂)_(n)NHC(O)—, —(CH₂)_(n)NHC(O)(CH₂)—,—NH(CH₂)_(n)C(O)—, —(CH₂)_(n)—, —(CH₂)_(n)(heterocyclyl)S(CH₂)_(n)C(O)—,and n is an integer from 1 to 10, and acid salts thereof.
 82. The methodof claim 51, wherein the linking group comprises an acyl or substitutedacyl group attached to the nitrogen atom.
 83. The method of claim 51,wherein the linking group comprises an alkyl or substituted alkyl groupattached to the nitrogen atom.
 84. The method of claim 51, wherein theantibody comprises: a variable heavy chain (V_(H)) polypeptidecomprising a V_(H) CDR1 comprising the amino acid sequence GFSLSSY (SEQID NO:2), a V_(H) CDR2 comprising the amino acid sequence DIKTGDR (SEQID NO:3), and a V_(H) CDR3 comprising the amino acid sequenceARVYVSGNDHYDL (SEQ ID NO:4); and a variable light chain (V_(L))polypeptide comprising a V_(L) CDR1 comprising the amino acid sequenceQSISNY (SEQ ID NO:6), a V_(L) CDR2 comprising the amino acid sequenceRAS (SEQ ID NO:7), and a V_(L) CDR3 comprising the amino acid sequenceQLGYTYSNVENA (SEQ ID NO:8).
 85. The method of claim 51, wherein theantibody comprises: a variable heavy chain (V_(H)) polypeptidecomprising a V_(H) CDR1 comprising the amino acid sequence GFSLSSY (SEQID NO:2), a V_(H) CDR2 comprising the amino acid sequence DIKTGDR (SEQID NO:3), and a V_(H) CDR3 comprising the amino acid sequenceARVYVSGNDHYDL (SEQ ID NO:4); and a variable light chain (V_(L))polypeptide comprising a V_(L) CDR1 comprising the amino acid sequenceQSISNY (SEQ ID NO:6), a V_(L) CDR2 comprising the amino acid sequenceRAS (SEQ ID NO:7), and a V_(L) CDR3 comprising the amino acid sequenceQLGYTYTNVENA (SEQ ID NO:10).
 86. The method of claim 51, wherein theantibody comprises: a variable heavy chain (V_(H)) polypeptidecomprising a V_(H) CDR1 comprising the amino acid sequence GFSFSSTK (SEQID NO:12), a V_(H) CDR2 comprising the amino acid sequence CIGTDT (SEQID NO:13), and a V_(H) CDR3 comprising the amino acid sequenceARSSSTGYYNL (SEQ ID NO:14); and a variable light chain (V_(L))polypeptide comprising a V_(L) CDR1 comprising the amino acid sequenceQSIRSY (SEQ ID NO:16), a V_(L) CDR2 comprising the amino acid sequenceYAS (SEQ ID NO:17), and a V_(L) CDR3 comprising the amino acid sequenceHDYYTFTDND (SEQ ID NO:18).
 87. The method of claim 51, wherein theantibody comprises: a variable heavy chain (V_(H)) polypeptidecomprising a V_(H) CDR1 comprising the amino acid sequence GFSFSSTK (SEQID NO:12); a V_(H) CDR2 comprising the amino acid sequence CIGVGSRGS(SEQ ID NO:20); and a V_(H) CDR3 comprising the amino acid sequenceARSSTTGYYIL (SEQ ID NO:21); and a variable light chain (V_(L))polypeptide comprising a V_(L) CDR1 comprising the amino acid sequenceESIYSY (SEQ ID NO:23); a V_(L) CDR2 comprising the amino acid sequenceKAS (SEQ ID NO:24); and a V_(L) CDR3 comprising the amino acid sequenceQNYYTFTEND (SEQ ID NO:25).
 88. The method of claim 51, wherein theantibody comprises: a variable heavy chain (V_(H)) polypeptidecomprising a V_(H) CDR1 comprising the amino acid sequence GFSFWR (SEQID NO:27); a V_(H) CDR2 comprising the amino acid sequence CIDGGNTNR(SEQ ID NO:28); and a V_(H) CDR3 comprising the amino acid sequenceARVRLGNNDYIDL (SEQ ID NO:29); and a variable light chain (V_(L))polypeptide comprising a V_(L) CDR1 comprising the amino acid sequenceQSISNY (SEQ ID NO:6); a V_(L) CDR2 comprising the amino acid sequenceRAS (SEQ ID NO:7); and a V_(L) CDR3 comprising the amino acid sequenceQQGYNWDLDGA (SEQ ID NO:31).
 89. The method of claim 84, wherein theantibody comprises: a variable heavy chain (V_(H)) polypeptidecomprising an amino acid sequence having 70% or greater identity to theamino acid sequence set forth in SEQ ID NO:1; and a variable light chain(V_(L)) polypeptide comprising an amino acid sequence having 70% orgreater identity to the amino acid sequence set forth in SEQ ID NO:5.90. The method of claim 85, wherein the antibody comprises: a variableheavy chain (V_(H)) polypeptide comprising an amino acid sequence having70% or greater identity to the amino acid sequence set forth in SEQ IDNO:1; and a variable light chain (V_(L)) polypeptide comprising an aminoacid sequence having 70% or greater identity to the amino acid sequenceset forth in SEQ ID NO:9.
 91. The method of claim 86, wherein theantibody comprises: a variable heavy chain (V_(H)) polypeptidecomprising an amino acid sequence having 70% or greater identity to theamino acid sequence set forth in SEQ ID NO:11; and a variable lightchain (V_(L)) polypeptide comprising an amino acid sequence having 70%or greater identity to the amino acid sequence set forth in SEQ IDNO:15.
 92. The method of claim 87, wherein the antibody comprises: avariable heavy chain (V_(H)) polypeptide comprising an amino acidsequence having 70% or greater identity to the amino acid sequence setforth in SEQ ID NO:19; and a variable light chain (V_(L)) polypeptidecomprising an amino acid sequence having 70% or greater identity to theamino acid sequence set forth in SEQ ID NO:22.
 93. The method of claim88, wherein the antibody comprises: a variable heavy chain (V_(H))polypeptide comprising an amino acid sequence having 70% or greateridentity to the amino acid sequence set forth in SEQ ID NO:26; and avariable light chain (V_(L)) polypeptide comprising an amino acidsequence having 70% or greater identity to the amino acid sequence setforth in SEQ ID NO:30.
 94. The method of claim 51, wherein the antibodyis a monoclonal antibody.
 95. The method of claim 94, wherein theantibody is a rabbit monoclonal antibody.
 96. The method of claim 51,wherein the antibody is selected from the group consisting of: an IgG,Fv, single chain antibody, scFv, Fab, F(ab′)2, and Fab′.
 97. The methodof claim 51, wherein the antibody is an IgG.
 98. The method of claim 97,wherein the antibody is an IgG1.
 99. The method of claim 51, wherein theantibody is a Fab.
 100. The method of claim 51, wherein the antibody isa single chain antibody.
 101. The method of claim 100, wherein theantibody is an scFv.
 102. The method of claim 51, wherein the antibodyis a polyclonal antibody.
 103. The method of claim 102, wherein theantibody is a rabbit polyclonal antibody.
 104. The method of claim 51,wherein the antibody further specifically binds to a metabolite ofsymmetric dimethylarginine.
 105. The method of claim 104, wherein themetabolite is symmetric Nα-acetyl-dimethylarginine (Ac-SDMA).